Increased Lung Epithelial Permeability Research Articles

Haploinsufficiency for Stard7 is associated with enhanced allergic responses in lung and skin.

Allergic asthma is a chronic inflammatory disorder that affects ∼20% of the population worldwide. Microarray analyses of nasal epithelial cells from acute asthmatic patients detected a 50% decrease in expression of Stard7, an intracellular phosphatidylcholine transport protein. To determine whether loss of Stard7 expression promotes allergic responses, mice were generated in which one allele of the Stard7 locus was globally disrupted (Stard7 (+/-) mice). OVA sensitization and challenge of Stard7(+/-) mice resulted in a significant increase in pulmonary inflammation, mucous cell metaplasia, airway hyperresponsiveness, and OVA-specific IgE compared with OVA-sensitized/challenged wild-type (WT) mice. This exacerbation was largely Th2-mediated with a significant increase in CD4(+)IL-13(+) T cells and IL-4, IL-5, and IL-13 cytokines. The loss of Stard7 was also associated with increased lung epithelial permeability and activation of proinflammatory dendritic cells in sensitized and/or challenged Stard7 (+/-) mice. Notably, OVA-pulsed dendritic cells from Stard7(+/-) mice were sufficient to confer an exaggerated allergic response in OVA-challenged WT mice, although airway hyperresponsiveness was greater in Stard7(+/-) recipients compared with WT recipients. Enhanced allergic responses in the lung were accompanied by age-dependent development of spontaneous atopic dermatitis. Overall, these data suggest that Stard7 is an important component of a novel protective pathway in tissues exposed to the extracellular environment.

Utility of urinary Clara cell protein (CC16) to demonstrate increased lung epithelial permeability in non-smokers exposed to outdoor secondhand smoke

The objective of this study was to assess the utility of urinary Clara cell protein (CC16) as a biomarker of increased lung epithelial permeability in non-smokers exposed to outdoor secondhand smoke. Twenty-eight healthy non-smoking adults visited outdoor patios of a restaurant and a bar where non-participants smoked and an open-air control with no smokers on three weekend days in a crossover study; subjects visited each site once for 3 h. Urine samples were collected at baseline, immediately post exposure and next morning, and analyzed for CC16. Changes in CC16 across location types or with cigarette count were analyzed using mixed-effect models, which included all subjects and stratified by gender. Urinary CC16 was higher in males (n=9) compared with females (n=18) at all measurement occasions (P<0.002), possibly reflecting prostatic contamination. Urinary CC16 from pre-exposure to post-exposure was higher following visits to restaurant and bar sites compared with the control among females but this increase did not reach statistical significance. Post-exposure to pre-exposure urinary CC16 ratios among females increased with cigarette count (P=0.048). Exposure-related increases in urinary CC16 were not seen among males. In conclusion, urinary CC16 may be a useful biomarker of increased lung epithelial permeability among female non-smokers; further work will be required to evaluate its applicability to males.

Cigarette smoking reprograms apical junctional complex molecular architecture in the human airway epithelium in vivo

The apical junctional complex (AJC), composed of tight and adherens junctions, maintains epithelial barrier function. Since cigarette smoking and chronic obstructive pulmonary disease (COPD), the major smoking-induced disease, are associated with increased lung epithelial permeability, we hypothesized that smoking alters the transcriptional program regulating airway epithelial AJC integrity. Transcriptome analysis revealed global down-regulation of physiological AJC gene expression in the airway epithelium of healthy smokers (n=59) compared to nonsmokers (n=53) in association with changes in canonical epithelial differentiation pathways such as PTEN signaling accompanied by induction of cancer-related AJC components. The overall expression of AJC-related genes was further decreased in COPD smokers (n=23). Exposure of airway epithelial cells to cigarette smoke extract in vitro resulted in down-regulation of several AJC genes paralleled by decreased transepithelial resistance. Thus, cigarette smoking induces transcriptional reprogramming of airway epithelial AJC architecture from its physiological pattern necessary for barrier function toward a disease-associated molecular phenotype.

Activation of endothelial cells after exposure to ambient ultrafine particles: The role of NADPH oxidase

Several studies have shown that ultrafine particles (UFPs) may pass from the lungs to the circulation because of their very small diameter, and induce lung oxidative stress with a resultant increase in lung epithelial permeability. The direct effects of UFPs on vascular endothelium remain unknown. We hypothesized that exposure to UFPs leads to endothelial cell O 2 − generation via NADPH oxidase and results in activation of endothelial cells. Our results showed that UFPs, at a non-toxic dose, induced reactive oxygen species (ROS) generation in mouse pulmonary microvascular endothelial cells (MPMVEC) that was inhibited by pre-treatment with the ROS scavengers or inhibitors, but not with the mitochondrial inhibitor, rotenone. UFP-induced ROS generation in MPMVEC was abolished by p67 phox siRNA transfection and UFPs did not cause ROS generation in MPMVEC isolated from gp91 phox knock-out mice. UFP-induced ROS generation in endothelial cells was also determined in vivo by using a perfused lung model with imaging. Moreover, Western blot and immunofluorescence staining results showed that MPMVEC treated with UFPs resulted in the translocation of cytosolic proteins of NADPH oxidase, p47 phox, p67 phox and rac 1, to the plasma membrane. These results demonstrate that NADPH oxidase in the pulmonary endothelium is involved in ROS generation following exposure to UFPs. To investigate the activation of endothelial cells by UFP-induced oxidative stress, we determined the activation of the mitogen-activated protein kinases (MAPKs) in MPMVEC. Our results showed that exposure of MPMVEC to UFPs caused increased phosphorylation of p38 and ERK1/2 MAPKs that was blocked by pre-treatment with DPI or p67 phox siRNA. Exposure of MPMVEC obtained from gp91 phox knock-out mice to UFPs did not cause increased phosphorylation of p38 and ERK1/2 MAPKs. These findings confirm that UFPs can cause endothelial cells to generate ROS directly via activation of NADPH oxidase. UFP-induced ROS lead to activation of MAPKs through induced phosphorylation of p38 and ERK1/2 MAPKs that may further result in endothelial dysfunction through production of cytokines such as IL-6. Our results suggest that endothelial oxidative stress may be an important mechanism for PM-induced cardiovascular effects.

CC16 as a marker of lung epithelial hyperpermeability in an acute model of rats exposed to mainstream cigarette smoke

The Clara cell secretory protein (CC16), which is produced along the tracheal-bronchial tree, has been shown to be a sensitive marker for the detection of lung hyperpermeability. Cigarette smoke inhalation has been associated with increased lung epithelial permeability. In this study we investigated the changes in CC16 in serum and bronchoalveolar lavage fluid (BALF) from female Sprague Dawley rats after a single exposure (2 × 1 h) to diluted mainstream cigarette smoke (MS) from the Reference Cigarette 2R4F. Rats were nose-only exposed to MS at concentrations of 0 (sham exposure), 250, 500, 750, 1000 or 1250 μg total particulate matter per liter. At 2, 4, 15 and 24 h after exposure, serum and BALF-samples were collected. CC16 was determined in BALF and serum. Albumin in BALF, another marker for lung permeability was also determined. A trend towards a lower CC16 recovery was observed in BALF from smoke-exposed rats. The CC16 concentration in serum showed a marked (up to five-fold) concentration- and time-dependent increase after MS exposure. The increase of CC16 in serum was most prominent at the early timepoints, i.e. 2 and 4 h after exposure, and a return to baseline concentrations was obvious at 24 h after exposure. The effect of MS exposure on the amount of albumin in BALF was limited (up to 60% increase). This study clearly showed that CC16 is a good marker for the assessment of the increased permeability of the lung/blood barrier after MS-exposure.

Clara cell protein and surfactant protein�B in garbage collectors and in wastewater workers exposed to bioaerosols

Inhalation of bioaerosols has been hypothesised to cause "toxic pneumonitis" that should increase lung epithelial permeability at the bronchioloalveolar level. Serum Clara cell protein (CC16) and serum surfactant protein B (SPB) have been proposed as sensitive markers of lung epithelial injury. This study was aimed at looking for increased lung epithelial permeability by determining CC16 and SPB in workers exposed to bioaerosols from wastewater or garbage. Subjects (778 wastewater, garbage and control workers; participation 61%) underwent a medical examination, lung function tests [American Thoracic Society (ATS) criteria], and determination of CC16 and SPB. Symptoms of endotoxin exposure and several potential confounders (age, gender, smoking, kidney function, obesity) were looked for. Results were examined with multiple linear or logistic regression. Exposure to bioaerosols increased CC16 concentration in the wastewater workers. No effect of exposure on SPB was found. No clue to work-related respiratory diseases was found. The increase in CC16 in serum supports the hypothesis that bioaerosols cause subclinical "toxic pneumonitis", even at low exposure.

Increased Serum and Urinary Concentrations of Lung Clara Cell Protein in Rats Acutely Exposed to Ozone

Clara cell protein (CC16) is a 16–17-kDa protein secreted by Clara cells in the bronchial lining fluid of the lung from which it passively diffuses into serum before being eliminated by the kidneys. The concentration of CC16 in serum has recently been proposed as a peripheral marker of the integrity of Clara cells and/or of the bronchoalveolar/blood barrier. To evaluate the sensitivity of this new lung marker to acute epithelial damage induced by ozone (O3), CC16 was determined in the serum of rats after a single 3-h exposure to 0.3, 0.6, or 1 ppm O3. The urinary excretion of the protein was also studied in rats repeatedly exposed to 1 ppm O3, 3 h/day, for up to 10 days. The concentrations of CC16 in the lung or trachea homogenates, the lung CC16 mRNA levels, and classical markers of lung injury in bronchoalveolar lavage fluid (BALF) were also determined. O3 produced a transient increase of CC16 concentration in serum that reached values on average 13 times above normal 2 h after exposure to 1 ppm O3. The intravascular leakage of CC16 was dose-dependent and correlated with the extent of lung injury as assessed by the levels of total protein, LDH, and inflammatory cells in BALF. This effect was most likely responsible for the concomitant marked reduction of CC16 concentrations in BALF and lung homogenate, since the CC16 mRNA levels in the lungs were unchanged and the absolute amounts of CC16 leaking into serum or lost from the respiratory tract were similar. These changes were paralleled by an elevation of the urinary excretion of CC16 resulting from an overloading of the tubular reabsorption process. These results demonstrate that the assay of CC16 in serum and even in urine represents a new noninvasive test to detect the increased lung epithelial permeability induced by O3.

Lung injury related to consuming Sauropus androgynus vegetable.

Taking Sauropus androgynus, a Malaysian food, to reduce weight began as a fad in Taiwan in 1994. Some advocates of this fad developed pulmonary dysfunction. The aim of this study is to report the lung injury in patients taking Sauropus androgynus. From July 1995 to November 1995, we investigated 104 nonsmoking patients (one male and 103 females) with chest roentgenography, pulmonary function, test, and Technetium 99m-labeled diethylene triamine penta-acetate (Tc-99m DTPA) radioaerosol inhalation lung scintigraphy. Among the 90 patients receiving Tc-99m DTPA inhalation lung scan, 46 (51.1%) patients had increased clearance of Tc-99m DTPA from lung and 20 (22.2%) patients had inhomogeneous deposition of the submicronic radioaerosol. Eighteen (18/100) patients had obstructive ventilatory impairment in pulmonary function test. Analyzing the results, we found that the patients with respiratory symptoms (n = 42) took more vegetables (p = 0.016), had increased clearance of Tc-99m DTPA (p = 0.010) and had lower FEV1 (p = 0.001), FEV1/FVC (p < 0.001), FEF25-75 (p = 0.001), VC (p = 0.002) and DLCO (p = 0.009) than the patients without respiratory symptoms (n = 62). FEV1 and FEV1/FVC were significantly reduced in patients with severe impairment of alveolar permeability. The cumulative dosage and duration of exposure were significantly associated with the reduction of FEV1 and FEV1/FVC. The lung injury after taking Sauropus androgynus involves alveoli and/or small airways and is manifest as obstructive ventilatory impairment with inhomogeneous aerosol distribution and increased lung epithelial permeability.

Alveolar endotoxin increases alveolar liquid clearance in rats.

Under some pathological conditions, ion transport across alveolar epithelial cells is downregulated, whereas under other pathological conditions, it may be upregulated. Because endotoxin is a biologically relevant pathological stimulus, we investigated the effect of endotoxin on alveolar epithelial liquid clearance in vivo. Escherichia coli endotoxin (220 micrograms/kg) was instilled into the lungs via the trachea of rats. Then, 24 or 40 h after endotoxin instillation, alveolar and lung liquid clearances were studied over 1 h by instillation of a 5% albumin solution with 1.5 microCi of 125I-labeled albumin (6 ml/kg into both lungs). Alveolar liquid clearance was significantly greater at 24 h (36 +/- 5%) and 40 h (38 +/- 7%) after endotoxin exposure than in saline-instilled controls (27 +/- 6%). Although there was an influx of neutrophils into the air space, there was no increase in lung epithelial permeability to protein at 24 or 40 h. Amiloride (2 x 10(-3) M), a sodium channel inhibitor, significantly reduced alveolar liquid clearance in the rats exposed to endotoxin. However, the increase in alveolar liquid clearance was not inhibited when propranolol (2 x 10(-5) M) was added to the 5% albumin solution. Thus exposure to alveolar endotoxin upregulates net alveolar fluid clearance in vivo for up to 40 h, a potentially important mechanism for accelerating alveolar fluid clearance under some pathological conditions. The increase in alveolar liquid clearance 24 and 40 h after instillation of endotoxin into the air spaces is mediated by an increased uptake of sodium through amiloride-sensitive sodium channels.

Vitamin E does not prevent exercise-induced increase in pulmonary clearance

It has been observed that sustained exercise results in a prolonged increase in alveolar epithelial permeability, as assessed by the pulmonary clearance rate of aerosolized 99mTc-labeled diethylenetriaminepentaacetate (Lorino et al. J. Appl. Physiol. 67: 2055-2059, 1989). The involvement of lipid peroxidation in this increased permeability was tested in seven nonsmoking volunteers by comparing the exercise-induced increases in pulmonary 99mTc-diethylenetriaminepentaacetate clearance before and after a 3-wk supplementation with oral vitamin E (1,000 IU/day), according to a protocol designed as a single-blind crossover study. The 60-min exercise was performed on a treadmill at a constant load corresponding to 80% of maximal O2 uptake. Administration of vitamin E, a very important antioxidant, did not reduce the exercise-induced increase in lung clearance, suggesting that the exercise-induced increase in lung epithelial permeability does not primarily result from the occurrence of lipid peroxidation in the alveolar membrane. This result thus corroborates the hypothesis of an alteration of the intercellular tight junctions due to the mechanical effects of hyperventilation.

An investigation of the role of glutathione in increased epithelial permeability induced by cigarette smoke in vivo and in vitro.

Airspace epithelial permeability is known to increase in cigarette smokers. To study the role of the antioxidant reduced glutathione (GSH) in this phenomenon, we used an in vitro model of the epithelial permeability of a monolayer of human type II alveolar epithelial cells (A549 cell line). Both whole (WSC) and vapor (VSC) smoke condensates induced a recoverable, concentration-dependent increase in epithelial permeability to 125iodine-labeled bovine serum albumin (125IBSA), associated with a profound fall in intracellular GSH. Buthionine sulfoximine (BSO), a GSH synthesis inhibitor, decreased GSH levels in A549 epithelial cells, significantly increased A549 epithelial cell permeability, and enhanced both WSC and VSC-induced A549 epithelial cell permeability. Co-culturing epithelial cells and GSH (500 microM) reduced WSC-induced, but not VSC-induced A549 epithelial cell permeability. Increasing intracellular GSH also ameliorated the smoke-induced increased epithelial permeability. Concentrations of cigarette smoke condensate of < 20% increased A549 epithelial cell permeability without associated cell detachment and lysis, which was also the case with BSO-induced increased epithelial permeability. WSC and VSC, instilled intratracheally, significantly increased rat lung epithelial permeability to 125IBSA, 6 h postinstillation, associated with a significant recruitment of neutrophils into the airspaces. This was associated with a small increase in GSH in the lung tissue of VSC-treated rats. However, both WSC and VSC markedly reduced GSH in bronchoalveolar lavage (BAL) fluid. Reduction in lung GSH to 95% but not to 68% of control values by BSO increased lung epithelial permeability in vivo. However, there was no additive effect on epithelial permeability of WSC and BSO.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of platelet-activating factor on lung epithelial permeability in the guinea-pig

We examined the effects of platelet-activating factor (PAF) on lung epithelial permeability by measuring the clearance of intratracheally administered 99m-technetium-labeled diethylene triamine penta-acetic acid ( 99mTc-DTPA) in guinea-pigs which were anaesthetised, paralysed and mechanically ventilated. The clearance of the radiolabeled tracer molecule 99mTc-DTPA from airways to the blood was expressed as changes in counts/min corrected for background. For each guinea-pig, 99mTc-DTPA clearance was assessed before and after i.v. PAF administration, when tracheal pressure had returned to near control values. Doses of 10, 50 and 100 ng/kg of PAF caused dose-dependent increases in 99mTc-DTPA clearance of 7 ± 3%, 38 ± 7% and 65 ± 11% respectively. The respective effects of 0.5 mg/kg of the beta 2-adrenergic agonist salbutamol and 0.3mg/kg of the alpha,-adrenergic agonist methoxamine on the increase in lung epithelial permeability induced by 50 ng/kg PAF were also studied. Salbutamol significantly reduced the acute bronchoconstrictor effects of PAF, but did not affect the increase in lung epithelial permeability, which was 58 ± 10%. Conversely, methoxamine significantly enhanced the broncho-constrictor effects of PAF but inhibited the lung epithelial permeability increase, which was only 10 ± 13%. In the absence of PAF, salbutamol significantly increased this permeability by 49 ± 11%, whereas methoxamine alone slightly reduced it, by −11 ± 4%. These results demonstrate that PAF increases lung epithelial permeability and suggest that vascular surface area recruitment may explain this increase.

Increased Lung Epithelial Permeability in HIV-infected Patients with Isolated Cytotoxic T-Lymphocytic Alveolitis

HIV-related lymphocytic alveolitis is common in HIV-seropositive patients without lung infection or tumor. In some of them a fraction of alveolar lymphocytes are HIV-specific cytotoxic T-lymphocytes (CTL) bearing the CD8 and D44 cell surface markers and capable of killing HIV-infected alveolar macrophages. In order to evaluate the in vivo effect of these CTL on lung function, we measured the pulmonary clearance of aerosolized 99mTc-diethylene triamine penta-acetate (DTPA-CI) on 24 occasions in 22 patients with lymphocytic alveolitis. DTPA-CI has been selected as a highly sensitive test to detect injury of the lung epithelium. In 13 of the patients, we found a high DTPA-CI of 4.56 +/- 2.54%.min-1 (mean +/- SD), suggesting an increase of the epithelial permeability. The lymphocytic alveolitis was then characterized by a high cellularity, a high proportion of lymphocytes (59 +/- 18%), mainly composed of CD8+D44+ T-lymphocytes (149 +/- 109 cells/mm3), which spontaneously exhibited a cytolytic activity against the autologous alveolar macrophages in a standard 51Cr release assay. In the remaining 11 patients, DTPA-CI was normal (less than 1.78%.min-1), lymphocytic alveolitis being characterized by a low number or an absence of CD8+D44+ alveolar lymphocytes (9 +/- 13 cells/mm3) with no significant cytolytic activity. In the whole group, a significant correlation (r = 0.74, p = 0.0004) was found between the DTPA-CI and the number of CD8+D44+ lymphocytes and their cytotoxic activity against alveolar macrophages. Altogether, these results suggest that an injury of the lung epithelium could result from a HIV-specific CTL-induced immunologic conflict.

Increased Lung Epithelial Permeability to Histamine is Mediated by H2 Receptors

Increased Lung Epithelial Permeability to Histamine is Mediated by H2 Receptors

HISTAMINE INCREASES LUNG PERMEABILITY BY AN H 2-RECEPTOR MECHANISM

The effect of specific H 1 and H 2 receptor antagonists on bronchial reactivity and increase in lung epithelial permeability in response to inhaled histamine was measured in 5 non-smoking men (age range 24-36 years). Inhaled histamine produced a short-lived but consistent increase in permeability to 99Tc-diethylenetriamine penta-acetate. An H 1-receptor antagonist, terfenadine (60 mg), protected against the bronchoconstrictor effect but had no significant influence on the increase in permeability. The H 2-receptor antagonist ranitidine (150 mg) significantly reduced the permeability response without having an effect on bronchial reactivity. These results demonstrate that the bronchoconstrictor effect of histamine is mediated by H 1 receptors and permeability increase is mediated by H 2 receptors. H 2-receptor mediated increase in lung epithelial permeability may be important clinically.

Pulmonary uptake of mannitol as an index of changes in lung epithelial permeability.

To develop a simple, inexpensive, and nontoxic test for changes in bronchial permeability we have employed a specific gas chromatographic assay to measure the uptake of mannitol from the lung after administration of an intratracheal aqueous bolus (8 ml of 5% solution). We have tested the ability of our method to detect the known increase in lung epithelial permeability elicited by challenge of previously sensitized animals with aerosols of Ascaris suum antigen. In Ascaris-sensitive Basenji-Greyhound dogs, antigen challenge significantly increased the rate of appearance and peak plasma level of mannitol after administration of the intratracheal bolus. Peak plasma mannitol levels averaged 25 +/- 3 (SE) micrograms/ml in dogs challenged with Ascaris antigen aerosols, and 14 +/- 2 micrograms/ml in the same animals in separate control experiments employing saline aerosols. The method presented could be readily applied to animal and human studies as a simple, inexpensive, and nontoxic indicator of lung epithelial permeability.