Search

Rationale: Modulation of the activity of sarcoendoplasmic reticulum calcium ATPase (SERCA) can profoundly affect Ca(2+) homeostasis. Although altered calcium homeostasis is a characteristic of cystic fibrosis (CF), the role of SERCA is unknown.Objectives: This study provides a comprehensive investigation of expression and activity of SERCA in CF airway epithelium. A detailed study of the mechanisms underlying SERCA changes and its consequences was also undertaken.Methods: Lung tissue samples (bronchus and bronchiole) from subjects with and without CF were evaluated by immunohistochemistry. Protein and mRNA expression in primary non-CF and CF cells was determined by Western and Northern blots.Measurements and Main Results: SERCA2 expression was decreased in bronchial and bronchiolar epithelia of subjects with CF. SERCA2 expression in lysates of polarized tracheobronchial epithelial cells from subjects with CF was decreased by 67% as compared with those from subjects without CF. Several non-CF and CF airway epithelial cell lines were also probed. SERCA2 expression and activity were consistently decreased in CF cell lines. Adenoviral expression of mutant F508 cystic fibrosis transmembrane regulator gene (CFTR), inhibition of CFTR function pharmacologically (CFTR(inh)172), or stable expression of antisense oligonucleotides to inhibit CFTR expression caused decreased SERCA2 expression. In CF cells, SERCA2 interacted with Bcl-2, leading to its displacement from caveolae-related domains of endoplasmic reticulum membranes, as demonstrated in sucrose density gradient centrifugation and immunoprecipitation studies. Knockdown of SERCA2 using siRNA enhanced epithelial cell death due to ozone, hydrogen peroxide, and TNF-alpha.Conclusions: Reduced SERCA2 expression may alter calcium signaling and apoptosis in CF. These findings decrease the likelihood of therapeutic benefit of SERCA inhibition in CF. [ABSTRACT FROM AUTHOR]

Rationale: Airway hyperresponsiveness is a critical feature of asthma. Substantial epidemiologic evidence supports a role for female sex hormones in modulating lung function and airway hyperresponsiveness in humans.Objectives: To examine the role of estrogen receptors in modulating lung function and airway responsiveness using estrogen receptor-deficient mice.Methods: Lung function was assessed by a combination of whole-body barometric plethysmography, invasive measurement of airway resistance, and isometric force measurements in isolated bronchial rings. M2 muscarinic receptor expression was assessed by Western blotting, and function was assessed by electrical field stimulation of tracheas in the presence/absence of gallamine. Allergic airway disease was examined after ovalbumin sensitization and exposure.Measurements and Main Results: Estrogen receptor-alpha knockout mice exhibit a variety of lung function abnormalities and have enhanced airway responsiveness to inhaled methacholine and serotonin under basal conditions. This is associated with reduced M2 muscarinic receptor expression and function in the lungs. Absence of estrogen receptor-alpha also leads to increased airway responsiveness without increased inflammation after allergen sensitization and challenge.Conclusions: These data suggest that estrogen receptor-alpha is a critical regulator of airway hyperresponsiveness in mice. [ABSTRACT FROM AUTHOR]

Rationale: Spleen tyrosine kinase (Syk) is important for Fc and B-cell receptor-mediated signaling. Objective: To determine the activity of a specific Syk inhibitor (R406) on mast cell activation in vitro and on the development of allergen-induced airway hyperresponsiveness (AHR) and inflammation in vivo. Methods: AHR and inflammation were induced after 10 d of allergen (ovalbumin [OVA]) exposure exclusively via the airways and in the absence of adjuvant. This approach was previously established to be IgE, FcepsilonRI, and mast cell dependent. Alternatively, mice were passively sensitized with OVA-specific IgE, followed by limited airway challenge. In vitro, the inhibitor was added to cultures of IgE-sensitized bone marrow-derived mast cells (BMMCs) before cross-linking with allergen. Results: The inhibitor prevented OVA-induced degranulation of passively IgE-sensitized murine BMMCs and inhibited the production of interleukin (IL)-13, tumor necrosis factor alpha, IL-2, and IL-6 in these sensitized BMMCs. When administered in vivo, R406 inhibited AHR, which developed in BALB/c mice exposed to aerosolized 1% OVA for 10 consecutive d (20 min/d), as well as pulmonary eosinophilia and goblet cell metaplasia. A similar inhibition of AHR was demonstrated in mice passively sensitized with OVA-specific IgE and exposed to limited airway challenge. Conclusion: This study delineates a functional role for Syk in the development of mast cell- and IgE-mediated AHR and airway inflammation, and these results indicate that inhibition of Syk may be a target in the treatment of allergic asthma. [ABSTRACT FROM AUTHOR]

Inhalation of powerful chemical agents, such as sulfur mustard (SM), can have debilitating pulmonary consequences, such as bronchiolitis obliterans (BO) and parenchymal fibrosis (PF). The underlying pathogenesis of disorders after SM inhalation is not clearly understood, resulting in a paucity of effective therapies. In this study, we evaluated the role of profibrotic pathways involving transforming growth factor-β (TGF-β) and platelet-derived growth factor (PDGF) in the development of BO and PF after SM inhalation injury using a rat model. Adult Sprague-Dawley rats were intubated and exposed to SM (1.0 mg/kg), then monitored daily for respiratory distress, oxygen saturation changes, and weight loss. Rats were killed at 7, 14, 21, or 28 days, and markers of injury were determined by histopathology; pulmonary function testing; and assessment of TGF-β, PDGF, and PAI-1 concentrations. Respiratory distress developed over time after SM inhalation, with progressive hypoxemia, respiratory distress, and weight loss. Histopathology confirmed the presence of both BO and PF, and both gradually worsened with time. Pulmonary function testing demonstrated a time-dependent increase in lung resistance, as well as a decrease in lung compliance. Concentrations of TGF-β, PDGF, and PAI-1 were elevated at 28 days in lung, BAL fluid, and/or plasma. Time-dependent development of BO and PF occurs in lungs of rats exposed to SM inhalation, and the elevated concentrations of TGF-β, PDGF, and PAI-1 suggest involvement of these profibrotic pathways in the aberrant remodeling after injury.

Sulfur mustard (bis 2-chloroethyl ethyl sulfide, SM) is a powerful bi-functional vesicating chemical warfare agent. SM tissue injury is partially mediated by the overproduction of reactive oxygen species resulting in oxidative stress. We hypothesized that using a catalytic antioxidant (AEOL 10150) to alleviate oxidative stress and secondary inflammation following exposure to SM would attenuate the toxic effects of SM inhalation. Adult male rats were intubated and exposed to SM (1.4 mg/kg), a dose that produces an LD 50 at approximately 24 h. Rats were randomized and treated via subcutaneous injection with either sterile PBS or AEOL 10150 (5 mg/kg, sc, every 4 h) beginning 1 h post-SM exposure. Rats were euthanized between 6 and 48 h after exposure to SM and survival and markers of injury were determined. Catalytic antioxidant treatment improved survival after SM inhalation in a dose-dependent manner, up to 52% over SM PBS at 48 h post-exposure. This improvement was sustained for at least 72 h after SM exposure when treatments were stopped after 48 h. Non-invasive monitoring throughout the duration of the studies also revealed blood oxygen saturations were improved by 10% and clinical scores were reduced by 57% after SM exposure in the catalytic antioxidant treatment group. Tissue analysis showed catalytic antioxidant therapy was able to decrease airway cast formation by 69% at 48 h post-exposure. To investigate antioxidant induced changes at the peak of injury, several biomarkers of oxidative stress and inflammation were evaluated at 24 h post-exposure. AEOL 10150 attenuated SM-mediated lung lipid oxidation, nitrosative stress and many proinflammatory cytokines. The findings indicate that catalytic antioxidants may be useful medical countermeasure against inhaled SM exposure., (© The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: Ozone pollution has adverse effects on respiratory health in children and adults. This study was carried out in the mouse model to investigate the influence of age and to define the role of toll-like receptor four (TLR4) in the lung response to ozone exposure during postnatal development., Methods: Female mice (1 to 6 weeks of age) were exposed for 3 h to ozone (1 part per million) or filtered air. Analyses were carried out at six and 24 h after completion of exposure, to assess the effects on lung permeability, airway neutrophilia, expression of antioxidants and chemokines, and mucus production. The role of TLR4 was defined by examining TLR4 expression in the lung during development, and by investigating the response to ozone in tlr4-deficient mice., Results: Metallothionein-1, calcitonin gene-related product, and chemokine C-X-C ligand (CXCL) five were consistent markers induced by ozone throughout development. Compared with adults, neonates expressed lower levels of pulmonary TLR4 and responded with increased mucus production, and developed an attenuated response to ozone characterized by reduced albumin leakage and neutrophil influx into the airways, and lower expression of CXCL1 and CXCL2 chemokines. Examination of the responses in tlr4-deficient mice indicated that ozone-mediated airway neutrophilia, but not albumin leakage or mucus production were dependent on TLR4., Conclusions: Collectively, the data demonstrate that the response to ozone is determined by age and is partially dependent on TLR4 signaling. The reduced responsiveness of the neonatal lung to ozone may be due at least in part to insufficient pulmonary TLR4 expression.

Autopsy specimens from human victims or experimental animals that die due to acute chlorine gas exposure present features of cardiovascular pathology. We demonstrate acute chlorine inhalation-induced reduction in heart rate and oxygen saturation in rats. Chlorine inhalation elevated chlorine reactants, such as chlorotyrosine and chloramine, in blood plasma. Using heart tissue and primary cardiomyocytes, we demonstrated that acute high-concentration chlorine exposure in vivo (500 ppm for 30 min) caused decreased total ATP content and loss of sarcoendoplasmic reticulum calcium ATPase (SERCA) activity. Loss of SERCA activity was attributed to chlorination of tyrosine residues and oxidation of an important cysteine residue, cysteine-674, in SERCA, as demonstrated by immunoblots and mass spectrometry. Using cardiomyocytes, we found that chlorine-induced cell death and damage to SERCA could be decreased by thiocyanate, an important biological antioxidant, and by genetic SERCA2 overexpression. We also investigated a U.S. Food and Drug Administration-approved drug, ranolazine, used in treatment of cardiac diseases, and previously shown to stabilize SERCA in animal models of ischemia-reperfusion. Pretreatment with ranolazine or istaroxime, another SERCA activator, prevented chlorine-induced cardiomyocyte death. Further investigation of responsible mechanisms showed that ranolazine- and istaroxime-treated cells preserved mitochondrial membrane potential and ATP after chlorine exposure. Thus, these studies demonstrate a novel critical target for chlorine in the heart and identify potentially useful therapies to mitigate toxicity of acute chlorine exposure.

Background: Inhalation of sulfur mustard (SM) and SM analog, 2-chloroethyl ethyl sulfide (CEES), cause fibrinous cast formation that occludes the conducting airways, similar to children with Fontan physiology-induced plastic bronchitis. These airway casts cause significant mortality and morbidity, including hypoxemia and respiratory distress. Our hypothesis was that intratracheal heparin, a highly cost effective and easily preserved rescue therapy, could reverse morbidity and mortality induced by bronchial cast formation., Methods: Sprague-Dawley rats were exposed to 7.5% CEES via nose-only aerosol inhalation to produce extensive cast formation and mortality. The rats were distributed into three groups: non-treated, phosphate-buffered saline (PBS)-treated, and heparin-treated groups. Morbidity was assessed with oxygen saturations and clinical distress. Blood and bronchoalveolar lavage fluid (BALF) were obtained for analysis, and lungs were fixed for airway microdissection to quantify the extent of airway cast formation., Results: Heparin, given intratracheally, improved survival (100%) when compared to non-treated (75%) and PBS-treated (90%) controls. Heparin-treated rats also had improved oxygen saturations, clinical distress and airway cast scores. Heparin-treated rats had increased thrombin clotting times, factor Xa inhibition and activated partial thromboplastin times, indicating systemic absorption of heparin. There were also increased red blood cells (RBCs) in the BALF in 2/6 heparin-treated rats compared to PBS-treated control rats., Conclusions: Intratracheal heparin 1 hr after CEES inhalation improved survival, oxygenation, airway obstruction, and clinical distress. There was systemic absorption of heparin in rats treated intratracheally. Some rats had increased RBCs in BALF, suggesting a potential for intrapulmonary bleeding if used chronically after SM inhalation., (© 2014 Wiley Periodicals, Inc.)

Rationale: Sulfur mustard (SM) is a chemical weapon stockpiled today in volatile regions of the world. SM inhalation causes a life-threatening airway injury characterized by airway obstruction from fibrin casts, which can lead to respiratory failure and death. Mortality in those requiring intubation is more than 80%. No therapy exists to prevent mortality after SM exposure. Our previous work using the less toxic analog of SM, 2-chloroethyl ethyl sulfide, identified tissue plasminogen activator (tPA) an effective rescue therapy for airway cast obstruction (Veress, L. A., Hendry-Hofer, T. B., Loader, J. E., Rioux, J. S., Garlick, R. B., and White, C. W. (2013). Tissue plasminogen activator prevents mortality from sulfur mustard analog-induced airway obstruction. Am. J. Respir. Cell Mol. Biol. 48, 439-447). It is not known if exposure to neat SM vapor, the primary agent used in chemical warfare, will also cause death due to airway casts, and if tPA could be used to improve outcome., Methods: Adult rats were exposed to SM, and when oxygen saturation reached less than 85% (median: 6.5 h), intratracheal tPA or placebo was given under isoflurane anesthesia every 4 h for 48 h. Oxygen saturation, clinical distress, and arterial blood gases were assessed. Microdissection was done to assess airway obstruction by casts., Results: Intratracheal tPA treatment eliminated mortality (0% at 48 h) and greatly improved morbidity after lethal SM inhalation (100% death in controls). tPA normalized SM-associated hypoxemia, hypercarbia, and lactic acidosis, and improved respiratory distress. Moreover, tPA treatment resulted in greatly diminished airway casts, preventing respiratory failure from airway obstruction., Conclusions: tPA given via airway more than 6 h after exposure prevented death from lethal SM inhalation, and normalized oxygenation and ventilation defects, thereby rescuing from respiratory distress and failure. Intra-airway tPA should be considered as a life-saving rescue therapy after a significant SM inhalation exposure incident., (© The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to model well differentiated human airway epithelium and heart muscle cells. This could be an invaluable tool to study the deleterious effects of toxic inhaled chemicals, such as chlorine, that can normally interact with the cell surfaces, and form various byproducts upon reacting with water, and limiting their effects in submerged cultures. Our model using well differentiated human airway epithelial cell cultures at air-liqiuid interface circumvents this limitation as well as provides an opportunity to evaluate critical mechanisms of toxicity of potential poisonous inhaled chemicals. We describe enhanced loss of membrane integrity, caspase release and death upon toxic inhaled chemical such as chlorine exposure. In this article, we propose methods to model chlorine exposure in mammalian heart and airway epithelial cells in culture and simple tests to evaluate its effect on these cell types.

Ozone pollution is associated with adverse effects on respiratory health in adults and children but its effects on the neonatal lung remain unknown. This study was carried out to define the effect of acute ozone exposure on the neonatal lung and to profile the transcriptome response. Newborn mice were exposed to ozone or filtered air for 3h. Total RNA was isolated from lung tissues at 6 and 24h after exposure and was subjected to microarray gene expression analysis. Compared to filtered air-exposed littermates, ozone-exposed newborn mice developed a small but significant neutrophilic airway response associated with increased CXCL1 and CXCL5 expression in the lung. Transcriptome analysis indicated that 455 genes were down-regulated and 166 genes were up-regulated by at least 1.5-fold at 6h post-ozone exposure (t-test, p < .05). At 24h, 543 genes were down-regulated and 323 genes were up-regulated in the lungs of ozone-exposed, compared to filtered air-exposed, newborn mice (t-test, p < .05). After controlling for false discovery rate, 50 genes were identified as significantly down-regulated and only a few (RORC, GRP, VREB3, and CYP2B6) were up-regulated at 24h post-ozone exposure (q < .05). Gene ontology enrichment analysis revealed that cell cycle-associated functions including cell division/proliferation were the most impacted pathways, which were negatively regulated by ozone exposure, an adverse effect that was associated with reduced bromo-deoxyuridine incorporation. These results demonstrate that acute ozone exposure alters cell proliferation in the developing neonatal lung through a global suppression of cell cycle function.

Sulfur mustard (SM) inhalation causes the rare but life-threatening disorder of plastic bronchitis, characterized by bronchial cast formation, resulting in severe airway obstruction that can lead to respiratory failure and death. Mortality in those requiring intubation is greater than 80%. To date, no antidote exists for SM toxicity. In addition, therapies for plastic bronchitis are solely anecdotal, due to lack of systematic research available to assess drug efficacy in improving mortality and/or morbidity. Adult rats exposed to SM analog were treated with intratracheal tissue plasminogen activator (tPA) (0.15-0.7 mg/kg, 5.5 and 6.5 h), compared with controls (no treatment, isoflurane, and placebo). Respiratory distress and pulse oximetry were assessed (for 12 or 48 h), and arterial blood gases were obtained at study termination (12 h). Microdissection of fixed lungs was done to assess airway obstruction by casts. Optimal intratracheal tPA treatment (0.7 mg/kg) completely eliminated mortality (0% at 48 h), and greatly improved morbidity in this nearly uniformly fatal disease model (90-100% mortality at 48 h). tPA normalized plastic bronchitis-associated hypoxemia, hypercarbia, and lactic acidosis, and improved respiratory distress (i.e., clinical scores) while decreasing airway fibrin casts. Intratracheal tPA diminished airway-obstructive fibrin-containing casts while improving clinical respiratory distress, pulmonary gas exchange, tissue oxygenation, and oxygen utilization in our model of severe chemically induced plastic bronchitis. Most importantly, mortality, which was associated with hypoxemia and clinical respiratory distress, was eliminated.

Tissue factor (TF) initiates the extrinsic coagulation cascade and is a high-affinity receptor for coagulation factor VII. TF also participates in protease-activated receptor (PAR)1 and PAR2 activation. Human epithelial basal cells were previously purified on the basis of TF expression. The purpose of this study was to determine if tracheobronchial epithelial basal cell-associated TF drives coagulation and/or activates PARs to promote basal cell functions. We used human tracheobronchial tissues to isolate human airway epithelial cells using specific cell surface markers by flow cytometry and studied TF expression by immunostaining. TF-dependent fibrin network formation was observed by confocal and scanning electron microscopy. TF knockdown was done using short hairpin RNA, and TF mRNA was measured using quantitative RT-PCR. We found that 97 ± 5% of first-passage human tracheobronchial epithelial cells were basal cells, and 100% of these basal cells expressed TF. Basal cell-associated TF was active, but TF activity was dependent on added extrinsic coagulation cascade factors. TF inhibition caused basal cell apoptosis and necrosis. This was due to two parallel but interdependent TF-regulated processes: failure to generate a basal cell-associated fibrin network and suboptimal PAR1 and PAR2 activity. The data indicate that membrane surface TF mediates airway epithelial basal cell attachment, which maintains cell survival and mitotic potential. The implications of these findings are discussed in the context of basal cell-associated TF activity in normal and injured tissues and of the potential for repair of airway epithelium in lung disease.

Background: Nanoparticles (NPs) produced by nanotechnology processes have taken the field of medicine by storm. Concerns about safety of these NPs in humans, however, have recently been raised. Although studies of NP toxicity have focused on lung disease the mechanistic link between NP exposure and lung injury remained unclear. This is primarily due to a lack of availability of appropriate airway disease models and sophisticated microscopic techniques to study nano-sized particulate delivery and resulting responses., Methods: Air-liquid interface (ALI) cultures of non-cystic fibrosis (CF) and CF airway epithelial cells were exposed to the FITC-labeled NPs using a PennCentury microsprayer™. Uptake of NPs was assessed by FACS. Laser scanning microscopy (LSM) was performed and the images were analyzed by an advanced imaging software to study particle deposition and uptake., Results: Flow cytometry data revealed that CF cells accumulated increased amounts of NPs. The increased NP uptake could be attributed to the reduced CF transmembrane conductance regulator (CFTR) function as a similar increased retention/uptake was observed in cells whose CFTR expression was downregulated by antisense oligonucleotide. NPs alone did not induce pro-inflammatory cytokine release or cell death. The cell culture system was sensitive to ozone but exposure to the uncoated synthetic NPs used in this study, did not cause any synergistic or suppressive effects. LSM imaging and subsequent image restoration further indicated particle uptake and intracellular localization. Exposure to ozone increased nuclear uptake in both non-CF and CF cells., Conclusion: Our findings demonstrate the uptake of NPs using ALI cultures of non-CF and CF airway epithelial cells. The NPs used here were useful in demonstrating uptake by airway epithelial cells without causing adverse effects in presence or absence of ozone. However, to totally exclude toxic effects, chronic studies under in vivo conditions using coated particulates are required.

The inhalation of sulfur mustard (SM) causes substantial deposition in the nasal region. However, specific injury has not been characterized. 2-chloroethyl ethyl sulfide (CEES) is an SM analogue used to model injury and screen potential therapeutics. After the inhalation of CEES, damage to the olfactory epithelium (OE) was extensive. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells were present by 4 hours, and maximal at 18-72 hours. Cleaved caspase 3 immunohistochemistry (IHC) was maximal at 18 hours after the inhalation of 5% CEES. Olfactory marker protein (OMP)-positive olfactory neurons were markedly decreased at 18 hours. IHC-positive cells for 3-nitrotyrosine (3-NT) within epithelium were elevated by 8 hours, waning by 18 hours, and absent by 72 hours. AEOL 10150, a catalytic manganoporphyrin antioxidant, administered both subcutaneously (5 mg/kg) and intranasally (50 μM, "combined treatment"), decreased OE injury. CEES-induced increases in markers of cell death were decreased by combined treatment involving AEOL 10150. CEES-induced changes in OMP and 3-NT immunostaining were markedly improved by combined treatment involving AEOL 10150. The selective inducible nitric oxide synthase inhibitor 1400W (5 mg/kg, subcutaneous), administered 1 hour after inhalation and thereafter every 4 hours (five doses), also reduced OE damage with improved OMP and 3-NT staining. Taken together, these data indicate that reactive oxygen and nitrogen species are important mediators in CEES-induced nasal injury.

Rationale: Sulfur mustard (SM) is a frequently used chemical warfare agent, even in modern history. SM inhalation causes significant respiratory tract injury, with early complications due to airway obstructive bronchial casts, akin to those seen after smoke inhalation and in single-ventricle physiology. This process with SM is poorly understood because animal models are unavailable., Objectives: To develop a rat inhalation model for airway obstruction with the SM analog 2-chloroethyl ethyl sulfide (CEES), and to investigate the pathogenesis of bronchial cast formation., Methods: Adult rats were exposed to 0, 5, or 7.5% CEES in ethanol via nose-only aerosol inhalation (15 min). Airway microdissection and confocal microscopy were used to assess cast formation (4 and 18 h after exposure). Bronchoalveolar lavage fluid (BALF) retrieval and intravascular dye injection were done to evaluate vascular permeability., Measurements and Main Results: Bronchial casts, composed of abundant fibrin and lacking mucus, occluded dependent lobar bronchi within 18 hours of CEES exposure. BALF contained elevated concentrations of IgM, protein, and fibrin. Accumulation of fibrin-rich fluid in peribronchovascular regions (4 h) preceded cast formation. Monastral blue dye leakage identified bronchial vessels as the site of leakage., Conclusions: After CEES inhalation, increased permeability from damaged bronchial vessels underlying damaged airway epithelium leads to the appearance of plasma proteins in both peribronchovascular regions and BALF. The subsequent formation of fibrin-rich casts within the airways then leads to airways obstruction, causing significant morbidity and mortality acutely after exposure.

The mechanism of ozone-induced lung cell injury is poorly understood. One hypothesis is that ozone induces lipid peroxidation and that these peroxidated lipids produce oxidative stress and DNA damage. Oxysterols are lipid peroxides formed by the direct effects of ozone on pulmonary surfactant and cell membranes. We studied the effects of ozone and the oxysterol 5beta,6beta-epoxycholesterol (beta-epoxide) and its metabolite cholestan-6-oxo-3,5-diol (6-oxo-3,5-diol) on human alveolar epithelial type I-like cells (ATI-like cells) and type II cells (ATII cells). Ozone and oxysterols induced apoptosis and cytotoxicity in ATI-like cells. They also generated reactive oxygen species and DNA damage. Ozone and beta-epoxide were strong inducers of nuclear factor erythroid 2-related factor 2, heat shock protein 70, and Fos-related antigen 1 protein expression. Furthermore, we found higher sensitivity of ATI-like cells compared to ATII cells exposed to ozone or treated with beta-epoxide or 6-oxo-3,5-diol. In general the response to the cholesterol epoxides was similar to the effect of ozone. Understanding the response of human ATI-like cells and ATII cells to oxysterols may be useful for further studies, because these compounds may represent useful biomarkers in other diseases., (Copyright 2010 Elsevier Inc. All rights reserved.)

Sulfur mustard (bis-2-(chloroethyl) sulfide; SM) is a highly reactive vesicating and alkylating chemical warfare agent. A SM analog, 2-chloroethyl ethyl sulfide (CEES), has been utilized to elucidate mechanisms of toxicity and as a screen for therapeutics. Previous studies with SM and CEES have demonstrated a role for oxidative stress as well as decreased injury with antioxidant treatment. We tested whether posttreatment with the metalloporphyrin catalytic antioxidant AEOL 10150 would improve outcome in CEES-induced lung injury. Anesthetized rats inhaled 5% CEES for 15 min via a nose-only inhalation system. At 1 and 9 h after CEES exposure, rats were given AEOL 10150 (5 mg/kg, sc). At 18 h post-CEES exposure BALF lactate dehydrogenase activity, protein, IgM, red blood cells, and neutrophils were elevated but were decreased by AEOL 10150 treatment. Lung myeloperoxidase activity was increased after CEES inhalation and was ameliorated by AEOL 10150. The lung oxidative stress markers 8-OHdG and 4-HNE were elevated after CEES exposure and significantly decreased by AEOL 10150 treatment. These findings demonstrate that CEES inhalation increased lung injury, inflammation, and oxidative stress, and AEOL 10150 was an effective rescue agent. Further investigation utilizing catalytic antioxidants as treatment for SM inhalation injury is warranted., ((c) 2010 Elsevier Inc. All rights reserved.)

Previous studies have shown that leukotriene B4 (LTB4), a proinflammatory lipid mediator, is linked to the development of airway hyperresponsiveness through the accumulation of IL-13-producing CD8+ T cells, which express a high affinity receptor for LTB4, BLT1 (Miyahara et al., Am J Respir Crit Care Med 2005;172:161-167; J Immunol 2005;174:4979-4984). By using leukotriene A4 hydrolase-deficient (LTA4H-/-) mice, which fail to synthesize LTB4, we determined the role of this lipid mediator in allergen-induced airway responses. Two approaches were used. In the first, LTA4H-/- mice and wild-type (LTA4H+/+) mice were systemically sensitized and challenged via the airways to ovalbumin. In the second, mice were passively sensitized with anti-ovalbumin IgE and exposed to ovalbumin via the airways. Mast cells were generated from bone marrow of LTA4H+/+ mice or LTA4H-/- mice. After active sensitization and challenge, LTA4H-/- mice showed significantly lower airway hyperresponsiveness compared with LTA4H+/+ mice, and eosinophil numbers and IL-13 levels in the bronchoalveoloar lavage of LTA4H-/- mice were also significantly lower. LTA4H-/- mice also showed decreased airway reactivity after passive sensitization and challenge. After LTA4H+/+ mast cell transfer, LTA4H-/- mice showed increased airway reactivity after passive sensitization and challenge, but not after systemic sensitization and challenge. These data confirm the important role for LTB4 in the development of altered airway responses and suggest that LTB4 secretion from mast cells is critical to eliciting increased airway reactivity after passive sensitization with allergen-specific IgE.

The female hormone estrogen is an important factor in the regulation of airway function and inflammation, and sex differences in the prevalence of asthma are well described. Using an animal model, we determined how sex differences may underlie the development of altered airway function in response to allergen exposure. We compared sex differences in the development of airway hyperresponsiveness (AHR) after allergen exposure exclusively via the airways. Ovalbumin (OVA) was administered by nebulization on 10 consecutive days in BALB/c mice. After methacholine challenge, significant AHR developed in male mice but not in female mice. Ovariectomized female mice showed significant AHR after 10-day OVA inhalation. ICI182,780, an estrogen antagonist, similarly enhanced airway responsiveness even when administered 1 hour before assay. In contrast, 17beta-estradiol dose-dependently suppressed AHR in male mice. In all cases, airway responsiveness was inhibited by the administration of a neurokinin 1 receptor antagonist. These results demonstrate that sex differences in 10-day OVA-induced AHR are due to endogenous estrogen, which negatively regulates airway responsiveness in female mice. Cumulatively, the results suggest that endogenous estrogen may regulate the neurokinin 1-dependent prejunctional activation of airway smooth muscle in allergen-exposed mice.

Phosphodiesterase IV (PDE IV) and neutral endopeptidase (NEP) may modulate the neurally mediated nonadrenergic noncholinergic inhibitory (NANCi) response. This response is not present in normal rabbits until 2 weeks of age. Allergen sensitization and challenge of fully grown 13-week old rabbits decreases the NANCi response. Our goal was to assess NANCi as a function of age and allergen sensitization. Tracheal smooth muscle (TSM) rings from normal 1-, 2-, and 13-week old rabbits plus ragweed immune as well as ragweed immune/challenged (I/C) 13-week old rabbits were assessed. Colorimetric assays of PDE IV and NEP activity were conducted on TSM from each group. NANCi responses were obtained in the presence and absence of Ro 20-1724 (PDE IV inhibitor) and/or phosphoramidon (Phos; NEP inhibitor) after contraction of TSM with neurokinin A. In normal TSM, there was no difference in PDE IV as a function of age. Conversely, NEP decreased significantly from 1 to 13 weeks of age. The immune and I/C groups had decreases in NEP and increases in PDE IV that were significant. Neither Ro 20-1724 nor Phos alone or together increased NANCi responses in TSM from 1- or 2-week old rabbits. However, both enhanced relaxation in TSM from normal, immune, and I/C 13-week old rabbits with an additive effect when drugs were combined. This work demonstrates (1) normal maturational changes in NEP but not PDE IV within TSM of this species; (2) modulation of the NANCi response by inhibitors of PDE IV and NEP in 13- but not 2-week old rabbits; (3) increased PDE IV and decreased NEP levels in the immune and I/C groups with reconstitution of NANCi responses by the combination of inhibitors. We conclude that mediation of the NANCi response is different in normal 2- and 13-week old rabbits. Both PDE IV and NEP modulated relaxation in fully grown rabbits, but had no effect at the younger age. Furthermore, both ragweed sensitization alone and ragweed challenge of immune rabbits altered NANCi via increases in PDE IV and decreases in NEP.

The mechanisms by which respiratory syncytial virus (RSV) infection causes airway hyperresponsiveness (AHR) are not fully established. We hypothesized that RSV infection may alter the expression of airway sensory neuropeptides, thereby contributing to the development of altered airway function. BALB/c mice were infected with RSV followed by assessment of airway function, inflammation, and sensory neuropeptide expression. After RSV infection, mice developed significant airway inflammation associated with increased airway resistance to inhaled methacholine and increased tracheal smooth muscle responsiveness to electrical field stimulation. In these animals, substance P expression was markedly increased, whereas calcitonin gene-related peptide (CGRP) expression was decreased in airway tissue. Prophylactic treatment with Sendide, a highly selective antagonist of the neurokinin-1 receptor, or CGRP, but not the CGRP antagonist CGRP(8-37), inhibited the development of airway inflammation and AHR in RSV-infected animals. Therapeutic treatment with CGRP, but not CGRP(8-37) or Sendide, abolished AHR in RSV-infected animals despite increased substance P levels and previously established airway inflammation. These data suggest that RSV-induced airway dysfunction is, at least in part, due to an imbalance in sensory neuropeptide expression in the airways. Restoration of this balance may be beneficial for the treatment of RSV-mediated airway dysfunction.

Sensory neuropeptides are localized to airway nerves and endocrine cells in both human and animal species and may participate in the development of airway inflammation and hyperresponsiveness (AHR). We used a mouse model to identify the changes that occur in calcitonin gene-related peptide (CGRP) expression in the airways during development of allergic inflammation and to investigate the potential role of this neuropeptide in modulating AHR. In sensitized mice, allergen challenge induced eosinophilic airway inflammation and AHR and resulted in considerable depletion of CGRP in neuroepithelial bodies and submucosal nerve plexuses without altering the overall density of airway nerve fibers. This depletion was subsequent to the development of airway inflammation and was prevented by anti-very late antigen-4 and anti-interleukin-5 treatments, which blocked airway eosinophilia and abolished AHR. Administration of CGRP to sensitized and challenged mice resulted in the normalization of airway responsiveness to inhaled methacholine, an effect that was neutralized by the receptor antagonist CGRP(8-37). These data demonstrate that replacement of CGRP following its depletion in allergic mice can reverse the changes in airway responsiveness and suggest that CGRP may have potential for the treatment of allergic AHR.

Within the respiratory epithelium of asthmatic patients, copper/zinc-containing superoxide dismutase (Cu/Zn SOD) is decreased. To address the hypothesis that lung Cu/Zn SOD protects against allergen-induced injury, wild-type and transgenic mice that overexpress human Cu/Zn SOD were either passively sensitized to ovalbumin (OVA) or actively sensitized by repeated airway exposure to OVA. Controls included nonsensitized wild-type and transgenic mice given intravenous saline or airway exposure to saline. After aerosol challenge to saline or OVA, segments of tracheal smooth muscle were obtained for in vitro analysis of neural control. In response to electrical field stimulation, wild-type sensitized mice challenged with OVA had significant increases in cholinergic reactivity. Conversely, sensitized transgenic mice challenged with OVA were resistant to changes in neural control. Stimulation of tracheal smooth muscle to elicit acetylcholine release showed that passively sensitized wild-type but not transgenic mice released more acetylcholine after OVA challenge. Function of the M(2) muscarinic autoreceptor was preserved in transgenic mice. These results demonstrate that murine airways with elevated Cu/Zn SOD were resistant to allergen-induced changes in neural control.

A dysfunction of pathways that normally cause contraction or relaxation of airways has been proposed to explain heightened levels of responsiveness produced by various insults to the airway. For example, we previously reported (4) that infection of cotton rats with the human respiratory syncytial virus (HRSV) leads to a significant decrease in an airway's nonadrenergic noncholinergic inhibitory (NANCi) response shortly after the infection. In the present study we addressed the more chronic effects of HRSV infection on airway function in young ferrets during a period of rapid somatic growth. Animals 1 wk old received HRSV or uninfected cell culture medium intranasally. In vitro studies of airway function were performed on tracheal smooth muscle (TSM) segments at 4, 8, and 24 wk of age. To evaluate neurally mediated contractile responses, frequency-response curves to electrical field stimulation (EFS) were performed with results expressed in terms of the frequency causing 50% of the maximal contractile response (ES50). In addition, contractile responses of TSM to methacholine (MCh) were also assessed with results expressed as the concentration needed to produce 50% of the maximal contractile response (EC50). To gauge NANCi responses, TSM was contracted with neurokinin A in the presence of atropine, propranolol, and indomethacin. Relaxant responses to EFS were assessed at frequencies from 5 to 30 Hz, with results expressed as mean percent relaxation. We found increased contractile responses to EFS in infected animals compared with that in the control group in both 4- and 8-wk old animals (p = 0.001 and p = 0.008, respectively). This difference had resolved by 24 wk of age. There was no difference in TSM responses to MCh between the groups at any age. Although there were no NANCi responses in 4-wk-old ferrets from either group, NANCi responses were significantly decreased in 8-wk-old ferrets previously infected with HRSV in the first week of life (p = 0.0001). A significant difference persisted (p = 0.008), albeit to a lesser degree, at 24 wk of age. These findings demonstrate that HRSV produces prolonged alterations of TSM function in ferret airways in vitro.

We studied the effects of recurrent aspiration of milk on neural control of airways in young developing rabbits. Beginning at 1 week of age, rabbits received 0.5 ml/kg of whole milk or sterile physiologic saline intranasally while under light methoxyflourane anesthesia 5 days a week for a period of 3 weeks. At 4 and 8 weeks of age, in vitro studies of contractile and relaxant responses of tracheal smooth muscle (TSM) segments were evaluated. To assess the neurally mediated contractile responses, frequency response curves to electrical field stimulation (EFS) were performed with results expressed in terms of frequency of EFS causing 50% of the maximal contractile response (ES50) values. In addition, the contractile responsiveness of TSM to methacholine (MCh) as reflected by the concentration causing 50% of the maximal contractile response (EC50) values was also determined to evaluate the underlying cholinergic reactivity of this segment of airway. To assess nonadrenergic noncholinergic inhibitory (NANCi) responses, experiments were performed on TSM contracted with neurokinin A in the presence of atropine, propranolol, and indomethacin. EFS was delivered to the contracted tissue at stimulation frequencies ranging from 5 to 30 Hz with results expressed as mean percent relaxation. Recurrent aspiration of milk but not saline increased EFS-induced contractile responses, as shown by significantly lower ES50 values compared with the control group: P = 0.02 and P = 0.001 at 4 and 8 weeks of age, respectively. TSM responsiveness to MCh was no different between the two groups, suggesting that alterations in prejunctional mechanisms of neural control were most likely responsible for the increased contractile response to EFS. The NANCi responses were significantly decreased by milk aspiration at both 4 and 8 weeks of age, with the abnormalities less pronounced at the later time point. These findings demonstrate that repeated aspiration of milk leads to abnormal mechanisms of neural control within airways of developing rabbits. While aspiration of milk altered both contractile and relaxant responses to EFS, the former abnormalities became more pronounced with time while the latter appeared to be resolving. These observations suggest that injury to an airway early in development does not necessarily resolve with time but may persist, with functional abnormalities becoming more pronounced even after the airway insult has ceased.

In a proportion of atopic asthmatics, exposure to a relevant antigen is followed by chronic inflammation in the airways leading to altered airway responsiveness (AR). However, the mechanisms underlying the development of airway hyperresponsiveness still remain unclear. To elucidate the relationship between IgE-mediated reactions and airway hyperresponsiveness, a murine model of passive sensitization and airway challenge with ovalbumin (OVA) was developed using anti-OVA IgE and IgG antibodies from murine B cell hybridomas. Passive sensitization by intravenous injection of anti-OVA IgE resulted in immediate cutaneous hypersensitivity and, after airway challenge with OVA on two consecutive days, increased AR in BALB/c and SJL mice. Increased numbers of eosinophils were observed in bronchoalveolar lavage fluid, in cells extracted from the lungs, and in the peribronchial areas of BALB/c mice passively sensitized with IgE and challenged through the airways compared with nonsensitized mice. Eosinophil peroxidase activity was also elevated in lung tissue from these mice. Passive sensitization with anti-OVA IgG1 but not IgG2a or IgG3 was similarly associated with development of skin test reactivity and increased AR after airway challenge, accompanied by an increase in eosinophils in bronchoalveolar lavage fluid. These data suggest that IgE/IgG1-mediated reactions together with local challenge with antigen can result in allergic inflammation resulting in altered airway function.

The effects of local versus systemic treatment with soluble IL-4 receptors (sIL-4R) were tested in a model of allergen-induced immediate hypersensitivity responses in BALB/c mice. Mice sensitized through the airways to ovalbumin (OVA) by ultrasonic nebulization once a week for 4 weeks developed increased serum anti-OVA IgE and IgG1 antibody titers and these were accompanied by immediate-type skin test responses to the allergen. These responses were also associated with the development of increased airway responsiveness (AR) as monitored by electrical field stimulation of tracheal smooth muscle preparations in vitro. Sensitized mice, treated by intraperitoneal injections of sIL-4R (150 micrograms/injection) administered in parallel to the sensitization protocol, developed significant suppression of anti-OVA IgE, anti-OVA IgG1 antibody production and of immediate cutaneous hypersensitivity responses. Airway responsiveness was normalized to some extent. Total IgE production was only slightly reduced. These effects were comparable to the findings following intraperitoneal injection of monoclonal anti-IL-4 antibody. Administration of sIL-4R via the airways was also effective in inhibiting the development of immediate hypersensitivity responses, including IgE production, and was more potent in normalizing airway responsiveness. These effects were achieved at lower concentrations than needed for systemic treatment. These data suggest that delivery of sIL-4R via the airways can effectively modulate the development of immediate hypersensitivity and airway hyperresponsiveness in response to aerosolized allergen.

The ability of subcutaneous pretreatment with an immunogenic peptide derived from Fel d I, the major cat protein, to suppress the development of allergic responses was examined in a mouse model of antigen-induced sensitization. BALB/c mice exposed to aerosolized Fel d I chain 1 peptide developed antigen-specific IgE responses, immediate cutaneous reactivity to the peptide, and increased airway responsiveness (AR). Both subcutaneous and intraperitoneal administration of the peptide prior to sensitization caused a 50% reduction in cutaneous reactivity which was associated with a decrease in serum anti-Fel d I chain 1 IgE and IgG1 antibody responses and an increase in specific IgG. Pretreatment with the peptide also suppressed spleen and lymph node proliferative responses to the peptide. However, only subcutaneous peptide injections could prevent the development of increased AR. Transfer of spleen cells from subcutaneously peptide-treated mice to sensitized recipients reduced serum antigen-specific IgE and IgG1 antibody responses and skin test reactivity, and prevented alterations in AR. These data suggest that IgE (and IgG1) responses and airway hyperresponsiveness induced by allergen sensitization via the airways can be modulated by subcutaneous administration of peptide. Further, the results define a model for investigating the modulatory effects of subcutaneous administration of immunogenic peptides or protein on an ongoing allergic response.

The role of allergen-specific sIgE+ B cells in the development of airway hyperresponsiveness to electrical field stimulation was examined in a murine model of allergic sensitization. Ovalbumin (OVA)-specific B cells (OVA+) were isolated from mice that were sensitized to aerosolized OVA. The OVA+ B cell population was shown to be distinct from the remaining, non-OVA-responsive B cells (OVA-). There was a high frequency of sIgE+ B cells and a low frequency of sIgG+ B cells in the OVA+ population compared with the OVA- population, where the ratio was reversed. Although both populations produced immunoglobulin in vitro, only the OVA+ cells secreted anti-OVA antibodies. Transfer of 10(6) OVA+ B cells or as few as 5 x 10(4) OVA+/sIgE+ B cells was able to transfer the capability for anti-OVA IgE synthesis and cutaneous reactivity to OVA in naive recipients. Exposure to OVA via the airways in addition to transfer of OVA+ B cells was necessary for development of airway hyperresponsiveness, whereas recipients challenged with an irrelevant allergen, ragweed, had normal airway function. Transfer of up to 10(7) OVA- B cells failed to induce production of anti-OVA IgE. Despite production of polyclonal IgE, recipients of OVA- B cells did not develop airway hyperresponsiveness after OVA challenge. We conclude that both allergen-specific IgE production and local challenge via the airways with specific allergen are necessary to change airway function in this model.

A dysfunction of the nonadrenergic noncholinergic inhibitory (NANCi) system has been invoked as a possible mechanism underlying or contributing to altered airway function. In the present study we assessed whether human respiratory syncytial virus (HRSV) infection affects the airways' neurally mediated contractile and relaxant (NANCi) responses in vitro. NANCi responses were studied on tracheal smooth muscle (TSM) segments obtained from young adult cotton rats, a well-established model for HRSV infection. To assess NANCi responses, TSM segments were removed and placed in tissue baths containing modified Krebs-Henseleit, atropine (1 x 10(-6) M) and propranolol (5 x 10(-6) M). After contraction with neurokinin A (1 x 10(-5) M), electrical field stimulation (EFS) was applied at stimulation frequencies ranging from 5 to 30 Hz. The NANCi responses were measured and expressed as the mean (+/- SE) percent relaxation. To evaluate neurally mediated contractile responses, full frequency response curves (0.5-30 Hz) to EFS were also performed. We found significantly decreased NANCi responses in TSM segments obtained from infected cotton rats (n = 12) compared with control animals (n = 9) (P < 0.002). Furthermore, the contractile responses to EFS were increased in infected animals compared with the control group (P = 0.0001). These findings demonstrate that HRSV infection leads to an enhanced contractile response to EFS and a significant decrease in NANCi response in cotton rat airways in vitro. This disruption of the neural control of airways may lead to the development of altered airway function.

We investigated the effects of substance P (SP) and vasoactive intestinal peptide (VIP) on acetylcholine (ACh) released from nerve endings by electrical field stimulation (EFS) in rabbit airways in vitro. ACh release was directly measured using high-performance liquid chromatography with electrochemical detection. Airway smooth muscle (ASM) segments, dissected from the midtrachea down to the left mainstem bronchus, were obtained from New Zealand White rabbits and mounted in organ baths containing modified Krebs-Henseleit solution, physostigmine, and choline. EFS at 20 Hz was delivered for 15 min to define baseline ACh release (pmol per gram of tissue per minute). There were no significant regional differences in ACh release during these baseline studies. A second stimulation was then performed in the absence (control) and presence of one or more of the following substances: SP (10(-7) M), a nonpeptide antagonist of the NK1 receptor (10(-7) M CP-96,345; Pfizer), and VIP (10(-7) M). Results for ACh release are expressed as a percentage of the first stimulation (means +/- SE). SP significantly increased ACh release in all ASM segments. This effect was abolished by CP-96,345. VIP alone did not affect ACh release. However, it significantly decreased SP-induced ACh release in all ASM segments. We conclude that SP significantly increases ACh release, thus facilitating cholinergic neurotransmission; its effect is abolished by CP-96,345. VIP decreases SP-induced ACh release, indicating a modulatory effect on cholinergic neurotransmission.

We studied the mechanisms involved in the airway smooth muscle (ASM) contraction to substance P (SP) in normal (control) and allergen-sensitized (immune) rabbits as well as immune rabbits exposed to allergen via the airways (immune challenged). Cumulative concentration-response curves to SP (1 x 10(-9) to 1 x 10(-4) M) were performed in ASM segments in the absence and presence of atropine (10(-5) M) in vitro. The maximal contractile response (g tension/g tissue) at 10(-4) M SP and ASM contractions at various concentrations of SP were expressed as means +/- SE. We found no difference in the contractile response to SP between control and immune animals. ASM segments obtained from immune-challenged rabbits were more responsive to SP. Atropine shifted to the right the concentration-response curves and decreased the maximal ASM contraction at 10(-4) M SP in all three groups; this effect, however, was greater in immune-challenged tissues. These findings demonstrate an increased contractile response to SP in immune-challenged animals mediated by a more pronounced facilitation of cholinergic neurotransmission. We conclude that the final ASM response to SP is the result of a complex interaction between direct effects on ASM and indirect effects through modulation of cholinergic neurotransmission.

We investigated the functional existence of the nonadrenergic noncholinergic inhibitory (NANCi) system in developing rabbit airways in vitro. Furthermore, we evaluated the effect of parenteral exposure to a specific allergen (ragweed) on the maturation of this neural pathway. NANCi responses were studied on tracheal smooth muscle (TSM) segments obtained from normal and ragweed-sensitized New Zealand White rabbits at 1, 2, 4, and 12 wk of age. The TSM segments were removed and placed in tissue baths containing modified Krebs-Henseleit solution, atropine (1 x 10(-5) M), and propranolol (5 x 10(-6) M). After contraction with neurokinin A (1 x 10(-5) M), electrical field stimulation was applied at stimulation frequencies ranging from 5 to 30 Hz to determine the frequency that produced maximal relaxation. The NANCi response to EFS was measured and expressed as the mean (+/- SE) percent relaxation at 20 Hz, because this stimulation frequency gave the maximal NANCi response at each age studied. TSM segments obtained from control rabbits at 1 wk of age did not demonstrate a NANCi response at the frequencies of stimulation used. By contrast, a reproducible NANC relaxation was demonstrated in TSM from 2-, 4-, and 12-wk-old rabbits. The magnitude of this response was 27 +/- 4.7 (n = 10), 29 +/- 4.8 (n = 9), and 37 +/- 4% (n = 18), respectively. The same experiments performed on TSM segments obtained from ragweed-sensitized animals gave significantly decreased values of NANCi response. In 2-, 4-, and 12-wk-old rabbits, the NANCi responses were 11.5 +/- 3.4 (n = 9), 11 +/- 2 (n = 13), and 16 +/- 4.2% (n = 14).(ABSTRACT TRUNCATED AT 250 WORDS)

A decrease in the airways' nonadrenergic noncholinergic inhibitory (NANC-i) system is one of the mechanisms that may contribute to allergen-induced changes in neural control within airways. We measured the airways' neurally mediated contractile and relaxant (NANC-i) responses in tracheal segments and left mainstem bronchus (LMB) from normal (control), immune (ragweed sensitized), and immune challenged rabbits. Immune rabbits were sensitized to mixed ragweed extract through parenteral injections from birth, while the immune challenged group had an additional airway exposure to aerosolized ragweed 48 hours prior to the in vitro studies. Neurally mediated contractile responses to electrical field stimulation (EFS) were increased in the immune challenged group, with the increase most significant in tracheal smooth muscle at a stimulation frequency of 20 Hz. To assess NANC-i responses, airway smooth muscle (ASM) segments from these groups were placed in tissue baths containing atropine (10(-6) M) and propranolol (5 x 10(-6) M). After contraction of the tissue with neurokinin A (NKA, 10(-5) M), the NANC-i response to EFS at 20 Hz was measured and reported as the mean (+/- SEM) percent relaxation. No significant differences were seen in the contractile responses of ASM segments to NKA among the three groups. The tracheal segments showed a significantly different NANC-i relaxation response among all groups: in the control group, 29.1 +/- 3.7; in the immune group 15.8 +/- 2.3%; and in the immune challenged group, 2.1 +/- 4.2%.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased release of acetylcholine (ACh) from airway parasympathetic nerve endings is one mechanism that may contribute to increases in airway responsiveness in immunoglobulin E (IgE)-immune allergen-exposed animals. We measured ACh released from murine tracheas following electrical field stimulation in vitro. BALB/c mice were immunized by exposure to an aerosol of 1% ovalbumin in sterile phosphate-buffered saline for 20 min/day for 10 days. At this time, levels of ovalbumin-specific IgE were proportionately higher than ovalbumin-specific IgG. As a control, nonimmune mice were similarly exposed to phosphate-buffered saline alone. Forty-eight hours after the last aerosol, tracheas were removed for assessment of either the contractile responses to electrical field stimulation and a cholinergic agonist (methacholine or ACh) or release of ACh produced by electrical field stimulation. ACh in the bath was measured using high-performance liquid chromatography with electrochemical detection. The stimulation frequencies causing one-half the maximal contractile response to electrical field stimulation were 4.1 +/- 0.2 and 2.8 +/- 0.2 Hz (P = 0.0001) for nonimmune and immune mice, respectively, whereas the molar concentrations of methacholine causing one-half of the maximal contractile response did not significantly differ. In addition, the dose-response curves of immune and nonimmune tracheas to ACh were superimposable. A significant increase in ACh release was demonstrated at both 10 and 20 Hz in tracheas from immune mice. ACh release (pmol.g tissue-1.min-1) from nonimmune and immune murine tracheas, respectively, were 140 +/- 8 and 205 +/- 22 (P = 0.013) at 10 Hz and 147 +/- 13 and 227 +/- 14 (P = 0.008) at 20 Hz.(ABSTRACT TRUNCATED AT 250 WORDS)

T and B cell responses after sensitization to ragweed (RW) were examined in a mouse model in which BALB/c mice were exposed to the allergen by ultrasonic nebulization. Sensitization resulted in the stimulation of an IgE anti-RW response and was paralleled by a rise in IgG1 anti-RW titers. Skin testing for immediate cutaneous hypersensitivity revealed the presence of allergic type I reactions to RW. Sensitization to RW in this way was also associated with the development of increased airways responsiveness as determined by electrical field stimulation of preparations of tracheal smooth muscle. Histologic examination of the airways and the lung indicated the presence of a mononuclear cell infiltrate in the mucosa and submucosa of the airways that was accompanied by an enlargement of local draining lymph nodes of the airways and the lung. T cell populations were analyzed for the frequency of V beta-expressing T cells. Such analysis indicated that RW sensitization stimulated the expression of V beta 8.1+, V beta 8.2+, and V beta 13+ T cells in the local lymphoid tissue and of V beta 8.1+, V beta 8.2+, V beta 8.3+, V beta 9+ and V beta 14+ T cells in the spleen. Co-culture of these T cell populations with RW-primed B cells indicated that in the presence of RW, V beta 8.2 T cells stimulated IgE and IgG1 production, whereas the other T cell populations showed a different stimulation profile for Ig isotypes and IgG subclasses. The transfer of V beta 8.2 T cells from sensitized but not from nonsensitized control mice stimulated an allergen-specific IgE and IgG1 response and increased airways responsiveness in naive recipients. These data provide additional support for the pivotal role of specific V beta-expressing T cell subpopulations in the stimulation of IgE/IgG1 production and increased airways responsiveness.

We have examined the effects of repeated exposure to antigen on airway responses to cholinergic stimulation in two inbred strains of mice that are similar in underlying cholinergic airway responsiveness, yet differ in their ability to produce IgE. Both BALB/c and SJL/J mice were repeatedly exposed to ovalbumin by inhalation for a 10-d period. While the BALB/c mice developed IgE antibody to this allergen, the SJL/J strain failed to mount an appreciable IgE response. In vitro assessments of the response of tracheal smooth muscle from saline exposed mice (controls) of both strains demonstrated responses to both methacholine and electrical field stimulation that were not significantly different between the strains. Following exposure to ovalbumin, the BALB/c strain developed a significant increase in their response to electrical field stimulation, while their response to methacholine was unaltered. In contrast, the in vitro responsiveness to these stimuli did not increase in SJL/J mice following similar exposure to inhaled nebulized ovalbumin. The passive transfer of cells from the peribronchial lymph nodes of ovalbumin-sensitized BALB/c mice into syngeneic nonimmune mice also led to increases in responsiveness of tracheal smooth muscle to electrical field stimulation. In contrast, transfer of cells from nonsensitized mice did not alter responsiveness. These results suggest that murine species capable of developing an IgE response to allergen also develop alterations in the neural control of their airways. Further, this alteration appears to be lymphocyte dependent, in that cells found within peribronchial lymph nodes following allergen exposure are capable of transferring this increase in responsiveness to nonimmune mice.

Previous reports have shown that 12-0-tetradecanoylphorbol-13-acetate can activate phospholipase D in human peripheral blood mononuclear cells as measured by an enzyme-catalyzed transphosphatidylation reaction (phosphatidylethanol formation). In the present study, the mononuclear cells were fractionated by two procedures to identify the responsive cells. Contrary to earlier suggestions, the results indicate that phorbol ester does not stimulate phospholipase D activity in normal lymphocytes. Thus, phosphatidylethanol was not produced by T lymphocytes (isolated by sheep erythrocyte rosette formation) or by a mixture of T and B lymphocytes (isolated by centrifugal elutriation). Under the same conditions, phorbol ester was able to activate phospholipse D in fractions that contained predominately monocytes. Preliminary experiments have further shown that phorbol ester does not induce phospholipase D activity in human T cell leukemic lines (MOLT-3, CEM, JURKAT, PEER) but can do so in some, but not all, B cell lines that have been infected with Epstein-Barr virus.

Research in this laboratory is currently focused on the biochemical and pharmacological properties of liposomes in which an otherwise water-soluble drug is anchored to the lipid bilayers via an appropriate non-polar residue. To this end, we have synthesized three (I-III) methotrexate (MTX) derivatives of dimyristoylphosphatidylethanolamine (DMPE) by conjugation of the alpha and/or gamma glutamyl carboxyl groups of the drug with the amino function of the phospholipid. These derivatives have been characterized analytically and chromatographically as MTX-gamma-DMPE (I), MTX-alpha-DMPE (II), and MTX-alpha, gamma-diDMPE (III). The corresponding glycerophosphorylethanolamine analogs have also been prepared and identified. The biological activity of these compounds (as inhibitors of in vitro cell proliferation and dihydrofolate reductase) is described in the following paper.

Methotrexate has been conjugated (amide bond) via either the alpha or gamma, or both alpha and gamma, glutamyl carboxyl groups to the amino function of dihexanoylphosphatidylethanolamine (C6C6PE) and 1-tetradecanoyl-2-hexanoylphosphatidylethanolamine (C14C6PE). These phospholipid prodrugs (either free or incorporated into liposomes) were compared with the corresponding ditetradecanoylphosphatidylethanolamine (C14C14PE) conjugates, some of whose properties have been described previously, for their ability to inhibit the proliferation of human leukemic cells (CEM/O) or cells derived therefrom (CEM/MTX) that are resistant to methotrexate because of a defective drug transport system. Regardless of chain length, the gamma conjugates were more effective than either the alpha or the alpha, gamma conjugates, in inhibiting growth of the parent cells, confirming initial experiments with mouse cells. Chain length had, however, a pronounced influence on the capacity of the various gamma derivatives to circumvent the transport defect. For example, CEM/MTX cells were 120-fold less susceptible than CEM/O cells to inhibition by either methotrexate or methotrexate-gamma-C6C6PE, whereas both cell lines were equally sensitive to methotrexate-gamma-C14C14PE. Although less potent than either of the foregoing, methotrexate-gamma-C14C6PE could partially by-pass the defective transport system. These results suggest that methotrexate-gamma-PE derivatives with appropriate acyl residues might be useful probes to investigate the mechanism by which phospholipids in general are able to traverse cell membranes.

This study compares the ability of methotrexate and liposomes, in which the drug is anchored to the lipid bilayers via methotrexate-gamma-dimyristoylphosphatidylethanolamine, to inhibit proliferation of human leukemic cells (CEM/O) and cells derived from this line that are resistant to methotrexate because of either a defective transport system (CEM/MTX cells) or elevated levels of dihydrofolate reductase (CEM/R1 cells). Whereas CEM/O and CEM/MTX cells show a 120-fold difference in their susceptibility to methotrexate (as measured by the incorporation of tritiated deoxyuridine into DNA), both lines are equally sensitive to the liposomes. In contrast, proliferation of CEM/MTX cells is not inhibited significantly by methotrexate-gamma-glycerophosphorylethanolamine (MTX-gamma-glyceroPE), the water-soluble analog of MTX-gamma-DMPE. Both the ability of the liposomes to circumvent the transport defect, and the inability of MTX-gamma-glyceroPE to do so, were anticipated on the basis of previous experiments which show that thiamine pyrophosphate could antagonize inhibition of mouse 3T3 and L1210 cell proliferation by methotrexate and MTX-gamma-glyceroPE, but not inhibition by liposomes. Human cells (CEM/O) behave similarly. The present experiments also suggest that liposomes prepared with MTX-gamma-DMPE can partially reverse the methotrexate resistance of CEM/R1 cells that is due to overproduction of the target enzyme.

Previous investigations have shown that untargeted liposomes, in which methotrexate is anchored to the lipid bilayers as methotrexate-gamma-dimyristoylphosphatidylethanolamine (methotrexate-gamma-DMPE), can inhibit in vitro cell proliferation. To test the possibility that this inhibition may involve extracellular metabolism of methotrexate-gamma-DMPE, we have degraded it chemically (dilute alkali) or enzymatically (phospholipase A2, phospholipase C, phospholipase C plus phosphatase), and assayed the products using human lymphoblastoid T cells or a subline that has a defective methotrexate transport system. Neither methotrexate-gamma-(1-myristoyl)-glycerophosphorylethanolamine, methotrexate-gamma-glycerophosphorylethanolamine, methotrexate-gamma-phosphorylethanolamine, nor methotrexate-gamma-ethanolamine resemble methotrexate-gamma-DMPE sensitized liposomes or the free derivative in their ability to block tritiated deoxyuridine incorporation into DNA. When added extracellularly, these putative metabolites manifest a higher ID50 concentration and/or, unlike the liposomes or unincorporated methotrexate-gamma-DMPE, utilize the methotrexate transport system to enter cells. Additionally, we have synthesized methotrexate-gamma-dihexadecylphosphatidylethanolamine and methotrexate-gamma-hexadecylphosphorylethanolamine, analogs of methotrexate-gamma-DMPE that cannot be hydrolyzed by phospholipases A2, C and D; liposomes prepared with these derivatives are markedly less potent cytotoxic agents than methotrexate-gamma-DMPE sensitized liposomes. All together, these results are consistent with the conclusion that methotrexate-gamma-DMPE must undergo intracellular metabolism to exert optimal inhibition; they also bear on possible mechanisms by which methotrexate-gamma-DMPE may enter cells.

Because of the sustained interest in liposomes as immunogens and vehicles for drug delivery, the present investigation was designed to reevaluate the iodoacetyl group as a means of binding sulfhydryl-containing substances to liposomes in thioether linkage, and to develop an alternative method by which liposomes with bound ligand can be conveniently and rapidly separated from free ligand. For the purpose of the first goal, we synthesized a homologous series of dimyristoylphosphatidylethanolamine (DMPE) derivatives in which the iodoacetyl (IA) function was separated from the phospholipid amino group by either 0, 1, or 2 aminoethylthioacetyl (AETA) spacers. Results show that liposomes prepared with IA-DMPE can not bind 125I-radiolabeled rabbit IgG which had been thiolated by reaction with S-acetylmercaptosuccinic anhydride. Significant IgG attachment was, however, obtained with liposomes containing either IA-AETA-DMPE or IA-(AETA)2-DMPE, and the amount bound was directly related to spacer length. In contrast, spacer length had no effect on the covalent binding of a low molecular weight hapten, N-dinitrophenylcysteine. Other parameters (incubation time, IgG concentration, density of IA-(AETA)2-DMPE, sulfhydryl inhibitors) were also examined. To achieve the second objective, biotinyl-(AETA)2-DMPE was incorporated into the same liposomal bilayers that contained the iodoacetylated derivatives. Thus, liposomes with bound ligand could be readily precipitated by avidin, and washed free of unreacted IgG by low speed centrifugation. Comparative experiments with liposomes containing biotinyl-DMPE revealed that spacer length also had a pronounced effect on the avidin precipitability of liposomes in the presence of proteins that may be non-covalently absorbed or covalently bound to the model membrane surface.

Liposomes, which were prepared with the three methotrexate (MTX)-dimyristoylphosphatidylethanolamine (DMPE) derivatives described in the preceding paper, were tested for their ability to block proliferation of mouse 3T3 and L1210 cells. Tritiated deoxyuridine incorporation into DNA could be completely inhibited by liposomes sensitized with MTX-DMPE I (MTX-gamma-DMPE). Under similar conditions, liposomes containing MTX-DMPE II (MTX-alpha-DMPE) and MTX-DMPE III (MTX-alpha, gamma-diDMPE) produced partial and no inhibition, respectively. These effects on cell growth were paralleled by the capacity of liposomes, prepared with each of the DMPE derivatives, to inhibit dihydrofolate reductase isolated from L1210 cells. Analogous experiments with the three corresponding glycerophosphorylethanolamine (glyceroPE) analogs also indicated that MTX-glyceroPE I was the most effective inhibitor of both cell proliferation and enzymatic activity. However, MTX-DMPE I sensitized liposomes apparently enter target cells as a consequence of phagocytosis, and not via the ubiquitous methotrexate transport system that is employed by MTX-glyceroPE I. For example, novel use of thiamine pyrophosphate showed that this compound had no influence on inhibition of cell proliferation due to liposomes, whereas thiamine pyrophosphate could completely antagonize the inhibitory effects of methotrexate and MTX-glyceroPE I. The results are discussed with reference to possible therapeutic advantages of these liposomes.

This investigation describes a new procedure for the preparation of immunogenic liposomes which circumvents the need to synthesize the N-(hapten)-substituted derivatives of phosphatidylethanolamine that were previously employed for this purpose. The method is based on the generation of liposomes containing the N-hydroxysuccinimide (NHS) esters of either palmitic acid, cholesteryl-hemisuccinate, or N-succinyl-phosphatidylethanolamine. Reaction of these preformed liposomes with a hapten that possesses a substitutable amino group (e.g., DNP-lysine) results in covalent attachment of the hapten to the lipid bilayers. As a consequence of this binding, the liposomes can elicit formation of hapten-specific plaque-forming cells in mice. The reliability of this procedure is indicated by the fact that these liposomes share the essential immunological properties of liposomes sensitized by incorporation of N-substituted phosphatidylethanolamine derivatives (e.g., DNP-Cap-PE). Thus, the magnitude of the response was found to be dependent on: (a) the presence of lipid A in the liposomes; (b) the phospholipid composition of the liposomes; (c) the distance separating the DNP determinant from the liposomal surface. Additional applications of liposomes, which contain the NHS esters, are indicated.