Lung protease/anti-protease network and modulation of mucus production and surfactant activity

Lung epithelium guarantees gas-exchange (performed in the alveoli) and protects from external insults (pathogens, pollutants…) present within inhaled air. Both functions are facilitated by secretions lining airway surface liquid, mucus (in the upper airways) and pulmonary surfactant (in the alveoli). Mucins, the main glycoproteins present within the mucus, are responsible for its rheologic properties and participate in lung defense mechanisms. In parallel, lung collectins are pattern recognition molecules present in pulmonary surfactant that also modulate lung defense. During chronic airways diseases, excessive protease activity can promote mucus hypersecretion and degradation of lung collectins and therefore contribute to the pathophysiology of these diseases. Importantly, secretion of local and systemic anti-proteases might be crucial to equilibrate the protease/anti-protease unbalance and therefore preserve the function of lung host defense compounds and airway surface liquid homeostasis. In this review we will present information relative to proteases able to modulate mucin production and lung collectin integrity, two important compounds of innate immune defense. One strategy to preserve physiological mucus production and collectin integrity during chronic airways diseases might be the over-expression of local ‘alarm’ anti-proteases such as SLPI and elafin. Interestingly, a cross-talk between lung collectins and anti-protease activity has recently been described, implicating the presence within the lung of a complex network between proteases, anti-proteases and pattern recognition molecules, which aims to keep or restore homeostasis in resting or inflamed lungs.     

The lung HETEs (and EETs) up

Arachidonic acid metabolites of the cyclooxygenase and lipoxygenase pathways have a variety of important lung functions. Recent observations indicate that cytochrome P-450 (P-450) monooxygenases are also expressed in the lung, localized to specific pulmonary cell types (e.g., epithelium, endothelium, and smooth muscle), and may modulate critical lung functions. This review summarizes recent data on the presence and biological activity of P-450-derived eicosanoids in the pulmonary vasculature and airways, including effects on pulmonary vascular and bronchial smooth muscle tone and airway epithelial ion transport. We hypothesize a number of potential functions of P-450-derived arachidonate metabolites in the lungs such as contribution to hypoxic pulmonary vasoconstriction, regulation of bronchomotor tone, control of the composition of airway lining fluid, and limitation of pulmonary inflammation. Finally, we describe a number of emerging technologies, including congenic and transgenic strains of experimental animals, P-450 isoform-specific inhibitors and inhibitory antibodies, eicosanoid analogs, and vectors for delivery of P-450 cDNAs and antisense oligonucleotides. These tools will facilitate further studies on the contribution of endogenously formed P-450 eicosanoid metabolites to lung function, under both normal and pathological conditions.     

Visualization of layers lining the lung of the South American lungfish (Lepidosiren paradoxa) and a comparison with the frog and rat.

Comarisons have been made of the structure of layers lining the lungs of lungfish, frog and rat using material fixed by perfusion of the pulmonary circulation of physiological pressures and at normal air pressures within the lung. The lining consists of a thin densely osmiophilic surface layer which covers a much thicker hypophase of generally floccular appearance. Tubular myelin, present in the frog and rat lung lining, was not observed in the layers lining the lung of Lepidosiren.     

Immunomodulatory mechanisms of lactobacilli

Over the past decade it has become clear that lactobacilli and other probiotic and commensal organisms can interact with mucosal immune cells or epithelial cells lining the mucosa to modulate specific functions of the mucosal immune system. The most well understood signalling mechanisms involve the innate pattern recognition receptors such as Toll-like receptors, nucleotide oligomerization domain-like receptors and C-type lectin receptors. Binding of microbe-associated molecular patterns with these receptors can activate antigen presenting cells and modulate their function through the expression of surface receptors, secreted cytokines and chemokines. In vitro the cytokine response of human peripheral blood mononuclear cells and dendritic cells to lactobacilli can be strikingly different depending on both the bacterial species and the strain. Several factors have been identified in lactobacilli that influence the immune response in vitro and in vivo including cell surface carbohydrates, enzymes modifying the structure of lipoteichoic acids and metabolites. In mice mechanistic studies point to a role for the homeostatic control of inducible T regulatory cells in the mucosal tissues as one possible immunomodulatory mechanism. Increasing evidence also suggests that induction of epithelial signalling by intestinal lactobacilli can modulate barrier functions, defensin production and regulate inflammatory signalling. Other probiotic mechanisms include modulation of the T cell effector subsets, enhancement of humoral immunity and interactions with the epithelial-associated dendritic cells and macrophages. A major challenge for the future will be to gain more knowledge about the interactions occurring between lactobacilli and the host in vivo and to understand the molecular basis of innate signalling in response to whole bacteria which trigger multiple signalling pathways.     

The chloride channel ClC-4 co-localizes with cystic fibrosis transmembrane conductance regulator and may mediate chloride flux across the apical membrane of intestinal epithelia.

Cystic fibrosis (CF) causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) lead to mislocalization of CFTR protein from the brush border membrane of epithelial tissues and/or its dysfunction as a chloride channel. In initial reports, it was proposed that certain channels from the ClC family of chloride channels may provide compensatory or alternative pathways for epithelial chloride secretion in tissues from cystic fibrosis patients. In the present work, we provide the first evidence that ClC-4 protein is functionally expressed on the surface of the intestinal epithelium and hence, is appropriately localized to act as a therapeutic target in this CF-affected tissue. We show using confocal and electron microscopy that ClC-4 co-localizes with CFTR in the brush border membrane of the epithelium lining intestinal crypts in mouse and human tissues. In Caco-2 cells, a cell line thought to model human enterocytes, ClC-4 protein is expressed on the cell surface and also partially co-localizes with EEA1 and transferrin, marker molecules of early and recycling endosomes, respectively. Hence, like CFTR, ClC-4 may cycle between the plasma membrane and endosomal compartment. Furthermore, we show that ClC-4 functions as a chloride channel on the surface of these epithelial cells as antisense ClC-4 cDNA expression reduced the amplitude of endogenous chloride currents by 50%. These studies provide the first evidence that ClC-4 is endogenously expressed and may be functional in the brush border membrane of enterocytes and hence should be considered as a candidate channel to provide an alternative pathway for chloride secretion in the gastrointestinal tract of CF patients.     

Inhibiting Glutathione Metabolism in Lung Lining Fluid as a Strategy to Augment Antioxidant Defense

Glutathione is abundant in the lining fluid that bathes the gas exchange surface of the lung. On the one hand glutathione in this extracellular pool functions in antioxidant defense to protect cells and proteins in the alveolar space from oxidant injury; on the other hand, it functions as a source of cysteine to maintain cellular glutathione and protein synthesis. These seemingly opposing functions are regulated through metabolism by gamma-glutamyl transferase (GGT, EC 2.3.2.2). Even under normal physiologic conditions, lung lining fluid (LLF) contains a concentrated pool of GGT activity exceeding that of whole lung by about 7-fold and indicating increased turnover of glutathione at the epithelial surface of the lung. With oxidant stress LLF GGT activity is amplified even further as glutathione turnover is accelerated to meet the increased demands of cells for cysteine. Mouse models of GGT deficiency confirmed this biological role of LLF GGT activity and revealed the robust expansiveness and antioxidant capacity of the LLF glutathione pool in the absence of metabolism. Acivicin, an irreversible inhibitor of GGT, can be utilized to augment LLF fluid glutathione content in normal mice and novel GGT inhibitors have now been defined that provide advantages over acivicin. Inhibiting LLF GGT activity is a novel strategy to selectively augment the extracellular LLF glutathione pool. The enhanced antioxidant capacity can maintain lung epithelial cell integrity and barrier function under oxidant stress.     

Internet-based course on pulmonary pathophysiology

A course of seven video lectures on pulmonary pathophysiology has been placed on the internet. This is a companion to the course on respiratory physiology available at http://meded.ucsd.edu/ifp/jwest/. That course dealt with normal respiratory physiology, and the new lectures are about the function of the diseased lung. The topics covered include pulmonary function tests, chronic obstructive pulmonary disease, asthma and localized airway obstruction, restrictive lung diseases, pulmonary vascular diseases, environmental or industrial lung diseases (with a short section on neoplastic and infectious diseases), and respiratory failure. Although it could be argued that PhD physiologists do not have a responsibility for teaching pathophysiology, collaborative teaching has become increasingly common in medical schools where, for example, a pulmonary block includes both normal respiratory physiology and some pulmonary pathophysiology. It is hoped that these lectures will be useful to physiologists in that setting.     

The cascade mechanism to explain ozone toxicity: The role of lipid ozonation products

Ozone is so reactive that it can be predicted to be entirely consumed as it passes through the first layer of tissue it contacts at the lung/air interface. This layer includes the lung lining fluid (tracheobronchial surface fluid and alveolar and small airway lining fluid) and, where the lung lining fluid is thin or absent, the membranes of the epithelial cells that line the airways. Therefore, the biochemical changes that follow the inhalation of ozone must be relayed into deeper tissue strata by a cascade of ozonation products. Lipid ozonation products (LOP) are suggested to be the most likely species to act as signal transduction molecules. This is because unsaturated fatty acids are present in the lipids in both the lung lining fluid and in pulmonary cell bilayers, and ozone reacts with unsaturated fatty acids to produce ozone-specific products. Further, lipid ozonation products are finite in number, have structures that are predictable from the Criegee ozonation mechanism, and are small, diffusible, stable (or metastable) molecules. Preliminary data show that individual LOP cause the activation of specific lipases, which trigger the release of endogenous mediators of inflammation.     

An infrared reflection-absorption spectroscopy study of the secondary structure in (KL4)4K,a therapeutic agent for respiratory distress syndrome,in aqueous monolayers with phospholipids

KLLLLKLLLLKLLLLKLLLLK (KL(4)) has been suggested to mimic some aspects of the pulmonary surfactant protein SP-B and has been tested clinically as a therapeutic agent for respiratory distress syndrome in premature infants [Cochrane, C. G., and Revak, S. D. (1991) Science 254, 566-568]. It is of obvious interest to understand the mechanism of KL(4) function as a guide for design of improved therapeutic agents. Attenuated total reflection (ATR) IR measurements have indicated that KL(4) is predominantly alpha-helical with a transmembrane orientation in lipid multilayers (1), a geometry quite different from the originally proposed peripheral membrane lipid interaction. However, the lipid multilayer model required for ATR may not be the best experimental paradigm to mimic the in vivo function of KL(4). In the current experiments, IR reflection-absorption spectroscopy (IRRAS) was used to evaluate peptide secondary structure in monolayers at the air/water interface, the physical state that best approximates the alveolar lining. In contrast to the ATR-IR results, KL(4) (2.5-5 mol %) films with either DPPC or DPPC/DPPG (7/3 mol ratio) adopted an antiparallel beta-sheet structure at all surface pressures studied > or =5 mN/m, including pressures physiologically relevant for lung function (40-72 mN/m). In contrast, in DPPG/KL(4) films, the dominant conformation was the alpha-helix over the entire pressure range, a possible consequence of enhanced electrostatic interactions. IRRAS has thus provided unique molecular structure information and insight into KL(4)/lipid interaction in a physiologically relevant state. A structural model is proposed for the response of the peptide to surface pressure changes.     

成体干细胞肺重建研究进展

成体干细胞来源广泛,无伦理争议,成为近几年的关注热点。研究表明以骨髓来源的间充质干细胞为代表的成体干细胞具有较强的多系分化潜能,可以广泛的参与包括肺在内的受损组织的修复与重建。在动物实验中已观察到,供体来源的成体干细胞可以定向分化为受损肺组织的多种功能细胞,并且有抑制纤维化等病变产生的能力。在本文中,回顾了近年来与肺损伤重建和疾病治疗相关的干细胞研究的最新进展,并探讨了成体干细胞治疗肺疾病与损伤的临床应用前景。     

Changes of the Cu and Zn contents in lung and liver in intestinal ischemic reperfusion and general ischemic reperfusion in rabbits

The changes of pulmonary and liver Cu-Zn contents were determined and evaluated in intestinal ischemic reperfusion (IIR) and general ischemic reperfusion (GIR) of rabbits. The contents of pulmonary Zn and liver Cu were found to be lower, and Cu/Zn ratio increased in lung tissue and decreased in liver tissue in IIR. The contents of pulmonary Zn were increased, and the contents of liver Cu were decreased; Cu/Zn ratio also decreased in both tissues in GIR. Pulmonary Cu and liver Zn contents were not changed in IIR and GIR. These results showed that lower or higher Zn in lung tissue and lower Cu in liver tissue were related to the acute tissue injury during IIR and GIR, suggesting that regulating the state of pulmonary Zn and liver Cu should be attempted during the prevention and treatment of both ischemic reperfusions.     

Compartmentalization of vascular endothelial growth factor to the epithelial surface of the human lung

BACKGROUND: Based on assessment of mRNA expression, the lung is a major site of expression of the vascular endothelial growth factor (VEGF) gene, largely from type II alveolar epithelial cells. With the knowledge that VEGF can function to induce vascular leak, we hypothesized that to protect the lung from pulmonary edema, the VEGF produced in the lung must be compartmentalized from the pulmonary endothelium, and thus must be compartmentalized to the surface of the respiratory epithelium. MATERIAL AND METHODS: To assess this hypothesis, we quantified the levels of VEGF in human respiratory epithelial lining fluid recovered by bronchoalveolar lavage from normal individuals. RESULTS: Strikingly, human respiratory epithelial lining fluid contains 11 +/- 5 ng/mL as quantified by ELISA, a 500-fold greater concentration than plasma (22 +/- 10 pg/mL, p < 0.0005). Western analysis of BAL fluid proteins showed the major VEGF isoform in respiratory epithelial lining fluid is VEGF165. CONCLUSIONS: With the knowledge that proteins of molecular mass like VEGF (34 to 46 kDa) slowly diffuse across the alveolar epithelium, it is likely that this high level "reservoir" of VEGF protein on the respiratory epithelial surface plays a role in normal lung endothelial biology. However, this compartmentalized VEGF reservoir may also be a "Damocles sword" poised to induce lung endothelial permeability in conditions of acute lung injury when the integrity of the alveolar epithelial barrier is breached.     

Ozone and the lung: a sensitive issue

Ozone is a powerful oxidant and toxic air pollutant. As a gaseous pollutant, its primary target tissue is the lung and breathing slightly elevated concentrations of ozone results in a range of respiratory symptoms. These include decreased lung function and increased airway hyper-reactivity in 10-20% of the healthy population. Moreover, those with conditions such as asthma and chronic obstructive pulmonary disease (COPD) generally experience an exacerbation of their symptoms. Together, these observations suggest that certain individuals are particularly susceptible to this oxidant gas. The primary goal of this review is to examine the basis of this increased sensitivity. Ozone is a highly reactive gas that is consumed by reactive processes on reaching the first interface in the lung, the lung lining fluid compartment. Reactions between ozone and antioxidants tend to dominate in this compartment and these are generally thought of as beneficial, or protective interactions. In those instances when ozone reacts with other substrates in lung lining fluid such as protein or lipid, secondary oxidation products arise which transmit the toxic signals to the underlying pulmonary epithelium. The rules that govern the balance between beneficial and detrimental interactions in the lung lining fluid compartment are not well established but these may contribute, in part, to sensitivity. On reaching the lung surface, secondary oxidation products arising from ozone initiate a number of cellular responses. These include cytokine generation, adhesion molecule expression and tight junction modification. Together, these responses lead to the influx of inflammatory cells to the lung in the absence of a pathogenic challenge. Moreover, lung permeability is increased and oedema develops. The nature and extent of these responses are variable and often not related within an individual. Thus, although an improved appreciation of the general mechanism of action of ozone has been attained in recent years, the basis for individual susceptibility is still unclear.     

An electron microscopic study of the acid mucosubstance lining the alveoli of hamster lung

Synopsis Hamster lung has been investigated by electron microscopy using colloidal iron oxide and Ruthenium Red stains. A continuous layer of acid mucosubstance has been demonstrated on the surface of the alveolar epithelial cells. The lining layer associated with the membranous pneumonocytes has been found to be consistently thinner than that covering the granular pneumonocytes and alveolar macrophages. Strong binding of stain has also been observed in osmiophilic membranous material which is thought to represent fragments of pulmonary surfactant. The susceptibility of the alveolar lining material to digestion by neuramidase suggests that it consists mainly of a sialomucin.     

Surfactant protein A (SP-A): the alveolus and beyond.

Surfactant protein A (SP-A) is the major protein component of pulmonary surfactant, a material secreted by the alveolar type II cell that reduces surface tension at the alveolar air-liquid interface. The function of SP-A in the alveolus is to facilitate the surface tension-lowering properties of surfactant phospholipids, regulate surfactant phospholipid synthesis, secretion, and recycling, and counteract the inhibitory effects of plasma proteins released during lung injury on surfactant function. It has also been shown that SP-A modulates host response to microbes and particulates at the level of the alveolus. More recently, several investigators have reported that pulmonary surfactant phospholipids and SP-A are present in nonalveolar pulmonary sites as well as in other organs of the body. We describe the structure and possible functions of alveolar SP-A as well as the sites of extra-alveolar SP-A expression and the possible functions of SP-A in these sites.     

Surfactant gene polymorphisms and interstitial lung diseases

Pulmonary surfactant is a complex mixture of phospholipids and proteins, which is present in the alveolar lining fluid and is essential for normal lung function. Alterations in surfactant composition have been reported in several interstitial lung diseases (ILDs). Furthermore, a mutation in the surfactant protein C gene that results in complete absence of the protein has been shown to be associated with familial ILD. The role of surfactant in lung disease is therefore drawing increasing attention following the elucidation of the genetic basis underlying its surface expression and the proof of surfactant abnormalities in ILD.     

Tumor Necrosis Factor-α +489G/A gene polymorphism is associated with chronic obstructive pulmonary disease

Pulmonary surfactant is a complex mixture of phospholipids and proteins, which is present in the alveolar lining fluid and is essential for normal lung function. Alterations in surfactant composition have been reported in several interstitial lung diseases (ILDs). Furthermore, a mutation in the surfactant protein C gene that results in complete absence of the protein has been shown to be associated with familial ILD. The role of surfactant in lung disease is therefore drawing increasing attention following the elucidation of the genetic basis underlying its surface expression and the proof of surfactant abnormalities in ILD.     

Tissue-level cytoprotection

In vitro and ex vivo tissue models provide a useful level of biological organization for cytoprotection studies positioned between cultured cells and intact animals. We have used 2 such models, primary tissue cultures of winter flounder renal secretory epithelium and ex vivo preparations of rat intestinal tissues, the latter to access the microcirculation of exposed mesentery tissues. Herein we discuss studies indicating that differentiated functions are altered in thermotolerant or cytoprotected tissues. These functions include transepithelial transport in renal epithelium and attachment and transmigration of leukocytes across vascular endothelium in response to mediators of inflammation. Evidence pointing to inflammation as a major venue for the heat shock response in vertebrates continues to mount. One such venue is wound healing. Heat shock proteins are induced early in wound responses, and some are released into the extracellular wound fluid where they appear to function as proinflammatory cytokines. However, within responding cells in the wound, heat shock proteins contribute to the acquisition of a state of cytoprotection that protects cells from the hostile environment of the wound, an environment created to destroy pathogens and essentially sterilize the wound. We propose that the cytoprotected state is an anti-inflammatory state that contributes to limiting the inflammatory response; that is, it serves as a brake on inflammation.     

Pulmonary vasoactivity of lung endocrine cell-related peptides

Lung endocrine-like cells are believed to contain three immunohistochemically distinct peptides: bombesin, calcitonin, and Leu-enkephalin. Because these peptides exhibit smooth muscle stimulatory or inhibitory activity in some tissues, it has been suggested that their release from endocrine-like cells may influence airway or pulmonary vascular smooth muscle tone. To determine whether lung endocrine cell-related peptides could exert a regulatory influence in the pulmonary circulation, we evaluated their ability to constrict or dilate the vasculature of isolated perfused rat lungs. Neither bombesin nor calcitonin exhibited any pulmonary vascular effects. However, Leu-enkephalin provoked dose-dependent pulmonary vasoconstriction. These results suggest that Leu-enkephalin released from lung endocrine-like cells could be involved with regulation of pulmonary vascular tone.