Murine Lung Slices Research Articles

Modulating pro-fibrotic macrophages using yeast beta-glucan microparticles prepared by Pressurized Gas eXpanded liquid (PGX) Technology®

Pro-fibrotic M2-like macrophages are widely implicated in the pathogenesis and progression of lung fibrosis due to their production of pro-fibrotic growth factors and cytokines. Yeast beta-glucan (YBG) microparticles have shown potential as immunomodulators that can convert macrophage polarization from a pro-fibrotic phenotype to an anti-fibrotic phenotype through the engagement of the Dectin-1 receptor. However, the processing conditions used to fabricate YBG microparticles can lead to unpredictable immunomodulatory effects. Herein, we report the use of Pressurized Gas eXpanded liquids (PGX) Technology® to fabricate YBG (PGX-YBG) microparticles with higher surface areas, lower densities, and smaller and more uniform size distributions compared to commercially available spray-dried YBGs. PGX-YBG is shown to activate Dectin-1 more efficiently in vitro while avoiding significant TLR 2/4 activation. Furthermore, PGX-YBG microparticles effectively modulate M2-like fibrosis-inducing murine and human macrophages into fibrosis-suppressing macrophages both in vitro as well as in ex vivo precision-cut murine lung slices, suggesting their potential utility as a therapeutic for addressing a broad spectrum of fibrotic end-point lung diseases.

Osteoprotegerin is an Early Marker of the Fibrotic Process and of Antifibrotic Treatment Responses in Ex Vivo Lung Fibrosis

BackgroundLung fibrosis is a chronic lung disease with a high mortality rate with only two approved drugs (pirfenidone and nintedanib) to attenuate its progression. To date, there are no reliable biomarkers to assess fibrosis development and/or treatment effects for these two drugs. Osteoprotegerin (OPG) is used as a serum marker to diagnose liver fibrosis and we have previously shown it associates with lung fibrosis as well.MethodsHere we used murine and human precision-cut lung slices to investigate the regulation of OPG in lung tissue to elucidate whether it tracks with (early) fibrosis development and responds to antifibrotic treatment to assess its potential use as a biomarker.ResultsOPG mRNA expression in murine lung slices was higher after treatment with profibrotic cytokines TGFβ1 or IL13, and closely correlated with Fn and PAI1 mRNA expression. More OPG protein was released from fibrotic human lung slices than from the control human slices and from TGFβ1 and IL13-stimulated murine lung slices compared to control murine slices. This OPG release was inhibited when murine slices were treated with pirfenidone or nintedanib. OPG release from human fibrotic lung slices was inhibited by pirfenidone treatment.ConclusionOPG can already be detected during the early stages of fibrosis development and responds, both in early- and late-stage fibrosis, to treatment with antifibrotic drugs currently on the market for lung fibrosis. Therefore, OPG should be further investigated as a potential biomarker for lung fibrosis and a potential surrogate marker for treatment effect.

Differential roles for lysyl oxidase (like), family members in chronic obstructive pulmonary disease; from gene and protein expression to function.

Chronic obstructive pulmonary disease (COPD) is characterized by long-term airflow obstruction with cigarette smoke as a key risk factor. Extracellular matrix (ECM) alterations in COPD may lead to small airway wall fibrosis. Altered collagen cross-linking, potentially mediated by the lysyl oxidase (LO) family of enzymes (LOX, LOXL1-4), orchestrates disturbed ECM homeostasis. In this study, we investigated the effects of smoking status and presence and severity of COPD on LOs gene and protein expression in the airways and the impact of LOs inhibition on airway contraction in an ex vivo mouse model. We used gene expression data from bronchial brushings, airway smooth muscle (ASM) cells in vitro and immunohistochemistry in lung tissue to assess smoke- and COPD-associated differences in LOs gene and protein expression in the small airways. We found higher LOX expression in current- compared to ex-smokers and higher LOXL1 expression in COPD compared to non-COPD patients. LOX and LOXL2 expression were upregulated in COPD ASM cells treated with cigarette smoke extract. LOXL1 and LOXL2 protein levels were higher in small airways from current- compared to non-smokers. In COPD patients, higher LOXL1 and lower LOX protein levels were observed, but no differences for LOXL2, LOXL3, and LOXL4 protein were detected in small airways. Inhibiting LOs activity increased airway contraction in murine lung slices. COPD-associated changes in LOs, in particular LOX and LOXL1, may be related to smoking and contribute to impaired airway function, providing potential novel targets for preventing or treating small airways changes in COPD.

Loss of ELK1 has differential effects on age-dependent organ fibrosis.

ETS domain-containing protein-1 (ELK1) is a transcription factor important in regulating αvβ6 integrin expression. αvβ6 integrins activate the profibrotic cytokine Transforming Growth Factor β1 (TGFβ1) and are increased in the alveolar epithelium in idiopathic pulmonary fibrosis (IPF). IPF is a disease associated with aging and therefore we hypothesised that aged animals lacking Elk1 globally would develop spontaneous fibrosis in organs where αvβ6 mediated TGFβ activation has been implicated. Here we identify that Elk1-knockout (Elk1-/0) mice aged to one year developed spontaneous fibrosis in the absence of injury in both the lung and the liver but not in the heart or kidneys. The lungs of Elk1-/0 aged mice demonstrated increased collagen deposition, in particular collagen 3α1, located in small fibrotic foci and thickened alveolar walls. Despite the liver having relatively low global levels of ELK1 expression, Elk1-/0 animals developed hepatosteatosis and fibrosis. The loss of Elk1 also had differential effects on Itgb1, Itgb5 and Itgb6 expression in the four organs potentially explaining the phenotypic differences in these organs. To understand the potential causes of reduced ELK1 in human disease we exposed human lung epithelial cells and murine lung slices to cigarette smoke extract, which lead to reduced ELK1 expression andmay explain the loss of ELK1 in human disease. These data support a fundamental role for ELK1 in protecting against the development of progressive fibrosis via transcriptional regulation of beta integrin subunit genes, and demonstrate that loss of ELK1 can be caused by cigarette smoke.

SiRNA-mediated protein knockdown in precision-cut lung slices

Small interfering RNA (siRNA) can induce RNA interference, which leads to the knockdown of messenger RNA (mRNA) and protein. As a result, siRNA is often used in vitro and in vivo to unravel the function of genes and as a therapeutic agent to disrupt excessive expression of disease-related genes. However, there is a large gap between in vitro and in vivo models in terms of simplicity, flexibility, throughput, and translatability. This gap could be bridged by using precision-cut tissue slices, which represent viable explants prepared from animal or human tissue that can be cultured ex vivo. Previously, we demonstrated that self-deliverable siRNA (Accell siRNA) induced significant mRNA knockdown in lung slices. The goal of this study, however, was to investigate whether Accell siRNA also induced protein knockdown in murine lung slices. Slices were incubated for up to 96 h with no siRNA (untransfected), non-targeting siRNA (control), or gene-targeting siRNA (Gapdh, Ppib, Serpinh1, and Bcl2l1). Overall, untransfected and transfected slices remained viable during an incubation of 96 h. In addition, gene-targeting siRNAs induced not only significant and specific mRNA knockdown but also protein knockdown. Finally, protein knockdown of fibrogenesis-related targets (Ppib, Serpinh1, and Bcl2l1) was shown to influence fibrogenesis on mRNA level, thereby demonstrating this model its utility in functional genomics and translational research.

Flagellar hook protein FlgE initiates a proinflammatory response via mixed pathways (MPF2P.762)

Abstract Among components of bacterial flagellum, filament flagellin C mediates immunostimulation via TLR5 pathway. Here we showed that hook protein FlgE also participates in host-pathogen interactions and stimulates innate immunity. Recombinant P. aeruginosa FlgE was obtained via routine prokaryotic expression system. After treatment of immortalized human corneal epithelial cells (HCEC) with FlgE, microarray profiling revealed that up-regulated genes were highly enriched in inflammation/immunity-related pathways. Inclusion of anti-TLR5 antibodies or 130mM KCl in culture each partially decreased the effects of FlgE, suggesting a mixed signaling pathways involvement. Pull-down assay performed on FlgE with HCEC lysates and following Mass Spectrum assay of bound proteins suggested that caveolin, membrane type ATP Synthase, Rab5 subunits and prohibitin might couple with FlgE. In an intranasal delivery model in mice as well in an organoid culture of murine lung slices, FlgE induced upregulation of IL1b, IL6 and CXCL1 at both mRNA and protein levels as detected by real-time PCR or ELISA. When administrated with ovalbumin in mice, FlgE enhanced OVA-specific CD4 T cells expansion. 3D modeling of FlgE disclosed two segments that would participate in FlgE polymerization during hook formation, and altering these two sites decreased immunostimulatory potency of FlgE. In conclusion, FlgE bears immunostimulation activity when confronting host cells, and might be used as an immunomodulator/adjuvant.

Selective targeting of the α5-subunit of GABAA receptors relaxes airway smooth muscle and inhibits cellular calcium handling.

The clinical need for novel bronchodilators for the treatment of bronchoconstrictive diseases remains a major medical issue. Modulation of airway smooth muscle (ASM) chloride via GABAA receptor activation to achieve relaxation of precontracted ASM represents a potentially beneficial therapeutic option. Since human ASM GABAA receptors express only the α4- and α5-subunits, there is an opportunity to selectively target ASM GABAA receptors to improve drug efficacy and minimize side effects. Recently, a novel compound (R)-ethyl8-ethynyl-6-(2-fluorophenyl)-4-methyl-4H-benzo[f]imidazo[1,5-a][1,4] diazepine-3-carboxylate (SH-053-2'F-R-CH3) with allosteric selectivity for α5-subunit containing GABAA receptors has become available. We questioned whether this novel GABAA α5-selective ligand relaxes ASM and affects intracellular calcium concentration ([Ca(2+)]i) regulation. Immunohistochemical staining localized the GABAA α5-subunit to human ASM. The selective GABAA α5 ligand SH-053-2'F-R-CH3 relaxes precontracted intact ASM; increases GABA-activated chloride currents in human ASM cells in voltage-clamp electrophysiology studies; and attenuates bradykinin-induced increases in [Ca(2+)]i, store-operated Ca(2+) entry, and methacholine-induced Ca(2+) oscillations in peripheral murine lung slices. In conclusion, selective subunit targeting of endogenous α5-subunit containing GABAA receptors on ASM may represent a novel therapeutic option to treat severe bronchospasm.

The omega-3 fatty acid docosahexaenoic acid attenuates organic dust-induced airway inflammation.

Workers exposed to organic dusts from concentrated animal feeding operations (CAFOs) are at risk for developing airway inflammatory diseases. Available preventative and therapeutic measures for alleviating dust-induced lung disease are inadequate. Because omega-3 fatty acids can mitigate inflammatory processes, we aimed to determine whether nutritional supplementation with the omega-3 fatty acid docosahexaenoic acid (DHA) could reduce the airway inflammatory consequences of exposures to organic dust. Aqueous extracts of organic dusts from swine CAFOs (ODE) were utilized. In DHA-pretreated human bronchial epithelial cells, lung fibroblasts, monocyte cell cultures, and precision-cut murine lung slices, we found that DHA pretreatment dose-dependently decreased ODE-induced inflammatory cytokine production. To determine the in vivo significance of DHA, C57BL/6 mice were orally administered DHA for seven days prior to treatment with intranasal ODE or saline inhalations. Animals treated with 2 mg DHA demonstrated significant reductions in ODE-induced bronchial alveolar lavage neutrophil influx and pro-inflammatory cytokine/chemokine production compared to mice exposed to ODE alone. Collectively, these data demonstrate that DHA affects several lung cells to reduce the airway inflammatory response to organic dust exposures. Dietary supplementation with DHA may be an effective therapeutic strategy to reduce the airway inflammatory consequences in individuals exposed to agriculture dust environments.

Architectural changes of the extracellular matrix in compensatory lung growth (540.2)

Compensatory growth of the remaining lung after murine pneumonectomy (PNX) involves the structural remodeling of the peripheral acini. Although the role of stem cells and local cellular proliferation is an area of active investigation, very little is known about the corresponding changes in the lung extracellular matrix (ECM). Post‐PNX adaptation of the elastin network, in particular, is essential for maintaining the mechanical and functional properties of the lung. Elastic fiber bundles concentrate particularly in the end bulbs of free septal alveolar edges shown in trans‐electron‐microscopic imaging and Verhoeff‐van‐Gieson staining. The elastic fibers form a continuous helical cable element, interconnecting alveolar entrance rings, spanning several levels of alveolar ducts. High‐resolution synchrotron radiation microscopy revealed this cord element running on the inside of alveolar ducts of infant murine lungs in the alveolar phase of lung development. The continuity of the elastic helix in adult c57bl/6 mice was visualized by scanning electron microscopy and by confocal microscopy of decellularized murine lung slices. Stereological estimates show an increased number of elastin‐containing septal edges during compensatory growth. The architectural changes in the murine ECM suggest active remodeling of the elastin network after PNX. The orientation of elastin in the septal tips of remodeling alveoli suggest the active participation of the elastin network in neoalveolarization. Consistent with these observations, the elastin cord element may be involved in the long‐range signaling essential for functional acinar remodeling.Grant Funding Source: NIH

Vitamin D Treatment Modulates Organic Dust–Induced Cellular and Airway Inflammatory Consequences

Exposure to organic dusts elicits airway inflammatory diseases. Vitamin D recently has been associated with various airway inflammatory diseases, but its role in agricultural organic dust exposures is unknown. This study investigated whether vitamin D reduces organic dust-induced inflammatory outcomes in cell culture and animal models. Organic dust extracts obtained from swine confinement facilities induced neutrophil chemokine production (human IL-8, murine CXCL1/CXCL2). Neutrophil chemokine induction was reduced in human blood monocytes, human bronchial epithelial cells, and murine lung slices pretreated with 1,25-(OH)(2) D(3) . Intranasal inhalation of organic dust extract induced neutrophil influx, and CXCL1/CXCL2 release was also decreased in mice fed a relatively high vitamin D diet as compared to mice fed a low vitamin D diet. These findings were associated with reduced tracheal epithelial cell PKCα and PKCε activity and whole lung TLR2 and TLR4 gene expression. Collectively, vitamin D plays a role in modulating organic dust-induced airway inflammatory outcomes.

Neuroepithelial Bodies as Mechanotransducers in the Intrapulmonary Airway Epithelium

In rodent lungs, a major part of the myelinated vagal airway afferents selectively contacts pulmonary neuroepithelial bodies (NEBs). Because most myelinated vagal airway afferents concern physiologically characterized mechanoreceptors, the present study aimed at unraveling the potential involvement of NEB cells in transducing mechanosensory information from the airways to the central nervous system. Physiological studies were performed using confocal Ca(2+) imaging of airway epithelium in murine lung slices. Mechanical stimulation by short-term application of a mild hypoosmotic solution (230 mosmol) resulted in a selective, fast, reversible, and reproducible Ca(2+) rise in NEB cells. Other airway epithelial cells could only be activated using more severe hypoosmotic stimuli (< 200 mosmol). NEB cells selectively expressed the Ca(2+)-permeable osmo- and mechanosensitive transient receptor potential canonical channel 5 (TRPC5) in their apical membranes, whereas immunoreactivity for TRP vanilloid-4 and TRP melastatin-3 was abundant in virtually all other airway epithelial cells. Hypoosmotic activation of NEB cells was prevented by GsMTx-4, an inhibitor of mechanosensitive ion channels, and by SKF96365, an inhibitor of TRPC channels. Short application of gadolinium, reported to activate TRPC5 channels, evoked a transient Ca(2+) rise in NEB cells. Osmomechanical activation of NEB cells gave rise to a typical delayed activation of Clara-like cells due to the release of ATP from NEB cells. Because ATP may activate the NEB-associated P2X(2/3) ATP receptor expressing myelinated vagal afferents, the current observations strongly suggest that pulmonary NEB cells are fully equipped to initiate mechanosensory signal transduction to the central nervous system via a purinergic signaling pathway.

IL-13Rα2 Reverses the Effects of IL-13 and IL-4 on Bronchial Reactivity and Acetylcholine-Induced Ca2+ Signaling

Background: The interleukins IL-4 and IL-13 play a key role in the pathophysiology of asthma. The interleukin receptor IL-13Rα2 is believed to act as a decoy receptor, but until now, the functional significance of IL-13Rα2 remains vague. Methods: Bronchial reactivity was quantified in murine lung slices by digital video microscopy and acetylcholine (ACH)-induced Ca²⁺ signaling was measured in human airway smooth muscle cells (ASMC) using fluorescence microscopy. Results: IL-4 or IL-13 up to 50 ng/ml induced bronchial hyperreactivity. But after incubation with 100 ng/ml this effect was lost and bronchial responsiveness was again comparable to the control level. The effects of IL-4 and IL-13 on bronchial reactivity were paralleled by the effects on ASMC proliferation. Fifty nanograms per milliliter of IL-4 and IL-13 increased the Ca²⁺ response of human ASMC to ACH. At 100 ng/ml, however, the effects of the cytokines on the Ca²⁺ response were no longer evident. The expression of IL-13Rα2 increased with increasing concentrations of IL-4 or IL-13, reaching its maximum at 100 ng/ml. Blocking IL-13Rα2, the loss of the effect of IL-4 and IL-13 at 100 ng/ml on human ASMC proliferation and the ACH-induced Ca²⁺ response were no longer present. Conclusions: IL-4 and IL-13 induce bronchial hyperreactivity by changing the Ca²⁺ homeostasis of ASMC. These effects are counteracted by IL-13Rα2. The biological significance of IL-13Rα2 might be a protective function by regulating IL-13- and IL-4-mediated signal transduction and thereby limiting pathological alterations in Th2-mediated inflammatory diseases.

Role of acetylcholine and polyspecific cation transporters in serotonin-induced bronchoconstriction in the mouse

BackgroundIt has been proposed that serotonin (5-HT)-mediated constriction of the murine trachea is largely dependent on acetylcholine (ACh) released from the epithelium. We recently demonstrated that ACh can be released from non-neuronal cells by corticosteroid-sensitive polyspecific organic cation transporters (OCTs), which are also expressed by airway epithelial cells. Hence, the hypothesis emerged that 5-HT evokes bronchoconstriction by inducing release of ACh from epithelial cells via OCTs.MethodsWe tested this hypothesis by analysing bronchoconstriction in precision-cut murine lung slices using OCT and muscarinic ACh receptor knockout mouse strains. Epithelial ACh content was measured by HPLC, and the tissue distribution of OCT isoforms was determined by immunohistochemistry.ResultsEpithelial ACh content was significantly higher in OCT1/2 double-knockout mice (42 ± 10 % of the content of the epithelium-denuded trachea, n = 9) than in wild-type mice (16.8 ± 3.6 %, n = 11). In wild-type mice, 5-HT (1 μM) caused a bronchoconstriction that slightly exceeded that evoked by muscarine (1 μM) in intact bronchi but amounted to only 66% of the response to muscarine after epithelium removal. 5-HT-induced bronchoconstriction was undiminished in M2/M3 muscarinic ACh receptor double-knockout mice which were entirely unresponsive to muscarine. Corticosterone (1 μM) significantly reduced 5-HT-induced bronchoconstriction in wild-type and OCT1/2 double-knockout mice, but not in OCT3 knockout mice. This effect persisted after removal of the bronchial epithelium. Immunohistochemistry localized OCT3 to the bronchial smooth muscle.ConclusionThe doubling of airway epithelial ACh content in OCT1/2-/- mice is consistent with the concept that OCT1 and/or 2 mediate ACh release from the respiratory epithelium. This effect, however, does not contribute to 5-HT-induced constriction of murine intrapulmonary bronchi. Instead, this activity involves 1) a non-cholinergic epithelium-dependent component, and 2) direct stimulation of bronchial smooth muscle cells, a response which is partly sensitive to acutely administered corticosterone acting on OCT3. These data provide new insights into the mechanisms involved in 5-HT-induced bronchoconstriction, including novel information about non-genomic, acute effects of corticosteroids on bronchoconstriction.

Airway smooth muscle relaxation results from a reduction in the frequency of Ca2+ oscillations induced by a cAMP-mediated inhibition of the IP3 receptor

BackgroundIt has been shown that the contractile state of airway smooth muscle cells (SMCs) in response to agonists is determined by the frequency of Ca2+ oscillations occurring within the SMCs. Therefore, we hypothesized that the relaxation of airway SMCs induced by agents that increase cAMP results from the down-regulation or slowing of the frequency of the Ca2+ oscillations.MethodsThe effects of isoproterenol (ISO), forskolin (FSK) and 8-bromo-cAMP on the relaxation and Ca2+ signaling of airway SMCs contracted with methacholine (MCh) was investigated in murine lung slices with phase-contrast and laser scanning microscopy.ResultsAll three cAMP-elevating agents simultaneously induced a reduction in the frequency of Ca2+ oscillations within the SMCs and the relaxation of contracted airways. The decrease in the Ca2+ oscillation frequency correlated with the extent of airway relaxation and was concentration-dependent. The mechanism by which cAMP reduced the frequency of the Ca2+ oscillations was investigated. Elevated cAMP did not affect the re-filling rate of the internal Ca2+ stores after emptying by repetitive exposure to 20 mM caffeine. Neither did elevated cAMP limit the Ca2+ available to stimulate contraction because an elevation of intracellular Ca2+ concentration induced by exposure to a Ca2+ ionophore (ionomycin) or by photolysis of caged-Ca2+ did not reverse the effect of cAMP. Similar results were obtained with iberiotoxin, a blocker of Ca2+-activated K+ channels, which would be expected to increase Ca2+ influx and contraction. By contrast, the photolysis of caged-IP3 in the presence of agonist, to further elevate the intracellular IP3 concentration, reversed the slowing of the frequency of the Ca2+ oscillations and relaxation of the airway induced by FSK. This result implied that the sensitivity of the IP3R to IP3 was reduced by FSK and this was supported by the reduced ability of IP3 to release Ca2+ in SMCs in the presence of FSK.ConclusionThese results indicate that the relaxant effect of cAMP-elevating agents on airway SMCs is achieved by decreasing the Ca2+ oscillation frequency by reducing internal Ca2+ release through IP3 receptors.

Modulation of the Ca2+ sensitivity of airway smooth muscle cells in murine lung slices

To investigate the phenomenon of Ca(2+) sensitization, we developed a new intact airway and arteriole smooth muscle cell (SMC) "model" by treating murine lung slices with ryanodine-receptor antagonist, ryanodine (50 microM), and caffeine (20 mM). A sustained elevation in intracellular Ca(2+) concentration ([Ca(2+)](i)) was induced in both SMC types by the ryanodine-caffeine treatment due to the depletion of internal Ca(2+) stores and the stimulation of a persistent influx of Ca(2+). Arterioles responded to this sustained increase in [Ca(2+)](i) with a sustained contraction. By contrast, airways responded to sustained high [Ca(2+)](i) with a transient contraction followed by relaxation. Subsequent exposure to methacholine (MCh) induced a sustained concentration-dependent contraction of the airway without a change in the [Ca(2+)](i). During sustained MCh-induced contraction, Y-27632 (a Rho-kinase inhibitor) and GF-109203X (a protein kinase C inhibitor) induced a concentration-dependent relaxation without changing the [Ca(2+)](i). The cAMP-elevating agents, forskolin (an adenylyl cyclase activator), IBMX (a phosphodiesterase inhibitor), and caffeine (also acting as a phosphodiesterase inhibitor), exerted similar relaxing effects. These results indicate that 1) ryanodine-caffeine treatment is a valuable tool for investigating the contractile mechanisms of SMCs while avoiding nonspecific effects due to cell permeabilization, 2) in the absence of agonist, sustained high [Ca(2+)](i) has a differential time-dependent effect on the Ca(2+) sensitivity of airway and arteriole SMCs, 3) MCh facilitates the contraction of airway SMCs by inducing Ca(2+) sensitization via the activation of Rho-kinase and protein kinase C, and 4) cAMP-elevating agents contribute to the relaxation of airway SMCs through Ca(2+) desensitization.

Role of Acetylcholine and Muscarinic Receptors in Serotonin-Induced Bronchoconstriction in the Mouse

For the murine trachea, it has been reported that constriction evoked by serotonin (5-HT) is largely dependent on acetylcholine (ACh) released from the epithelium, owing to the sensitivity of the 5-HT response to epithelium removal, sensitivity to atropine, and insensitivity to tetrodotoxin (Moffatt et al., 2003). Consistent with this assumption, the respiratory epithelium contains ACh, its synthesizing enzyme, and the high-affinity choline transporter CHT1 (Reinheimer et al., 1996; Pfeil et al., 2003; Proskocil et al., 2004). Recently, we demonstrated that ACh can be released from non-neuronal cells by corticosteroid-sensitive polyspecific organic cation transporters (OCTs), which are also expressed by airway epithelial cells (Lips et al., 2005). Hence, we proposed that 5-HT evokes release of ACh from epithelial cells via OCTs and that this epithelial-derived ACh induces bronchoconstriction. We tested this hypothesis in a well-established model of videomorphometric analysis of bronchial diameter in precision-cut murine lung slices utilizing epithelium removal to assess the role of the epithelium, OCT mouse knockout (KO) strains to assess the role of OCT isoforms, and muscarinic receptor M2/M3 double-KO mice to assess the cholinergic component of 5-HT induced bronchoconstriction, as bronchi of this strain are entirely unresponsive to cholinergic stimulation(Struckmann et al., 2003).

Acetylcholine-induced calcium signaling and contraction of airway smooth muscle cells in lung slices.

The Ca(2+) signaling and contractility of airway smooth muscle cells (SMCs) were investigated with confocal microscopy in murine lung slices (approximately 75-microm thick) that maintained the in situ organization of the airways and the contractility of the SMCs for at least 5 d. 10--500 nM acetylcholine (ACH) induced a contraction of the airway lumen and a transient increase in [Ca(2+)](i) in individual SMCs that subsequently declined to initiate multiple intracellular Ca(2+) oscillations. These Ca(2+) oscillations spread as Ca(2+) waves through the SMCs at approximately 48 microm/s. The magnitude of the airway contraction, the initial Ca(2+) transient, and the frequency of the subsequent Ca(2+) oscillations were all concentration-dependent. In a Ca(2+)-free solution, ACH induced a similar Ca(2+) response, except that the Ca(2+) oscillations ceased after 1--1.5 min. Incubation with thapsigargin, xestospongin, or ryanodine inhibited the ACH-induced Ca(2+) signaling. A comparison of airway contraction with the ACH-induced Ca(2+) response of the SMCs revealed that the onset of airway contraction correlated with the initial Ca(2+) transient, and that sustained airway contraction correlated with the occurrence of the Ca(2+) oscillations. Buffering intracellular Ca(2+) with BAPTA prohibited Ca(2+) signaling and airway contraction, indicating a Ca(2+)-dependent pathway. Cessation of the Ca(2+) oscillations, induced by ACH-esterase, halothane, or the absence of extracellular Ca(2+) resulted in a relaxation of the airway. The concentration dependence of the airway contraction matched the concentration dependence of the increased frequency of the Ca(2+) oscillations. These results indicate that Ca(2+) oscillations, induced by ACH in murine bronchial SMCs, are generated by Ca(2+) release from the SR involving IP(3)- and ryanodine receptors, and are required to maintain airway contraction.

Acute pneumocyte injury, poly(ADP-ribose) polymerase activity, and pyridine nucleotide levels after in vitro exposure of murine lung slices to cyclophosphamide

Cyclophosphamide (CYC) is a metabolically activated, DNA-alkylating, antitumor agent that causes pulmonary fibrosis. BALB/cN (B) mice are sensitive and C57B1/6N (C) mice are resistant to CYC-induced fibrosis. Pulmonary bioactivation may contribute to strain sensitivity. Therefore, we tested the intrinsic susceptibility of murine lung slices to cell injury by direct exposure to CYC for 2–8 hr. Injury was measured by release of lactate dehydrogenase (LDH). DNA damage activates the nuclear enzyme poly(ADP-ribose) polymerase (PAP, EC 2.4.2.30), causing depletion of its substrate, NAD. NAD can also be decreased by phosphorylation to NADP, as seen with oxidative stress. Depletion of NAD can lead to loss of ATP. Thus, we measured LDH release, PAP activation, NAD, NADP and ATP in slices incubated with or without the PAP-inhibitor, 3-aminobenzamide (3-AB). CYC (0.1 to 1.0 mg/mL for 4–8 hr) caused LDH release in slices from both murine strains, but LDH release was significantly greater in B lung slices than in C slices. After an 8-hr incubation 63.9±3.7% (mean±SEM) of total LDH was released from B lung slices with 1.0 mg CYC/mL, whereas only 45.8±2.6% was released from C lung slices (P<0.05). 3-AB reduced LDH release to 44.7±2.4% in B slices and 28.1±2.0% in C slices (P<0.05 vs CYC only). PAP activity in nuclei isolated from CYC-treated B lung slices was increased 2- to 4-fold after 2 hr of incubation with 0.5 and 1.0 mg CYC/mL. PAP activation was delayed and reduced with incubation in 3-AB. PAP was activated 2-fold in nuclei from C slices treated with 0.5 mg CYC/mL for 2 hr. NAD was decreased at 2 and 24 hr in B slices treated with 0.5 and 1.0 mg CYC/mL, and at 4 hr with 0.1 mg CYC/mL. NAD depletion occurred only at 4 hr in the resistant C slices treated with 1.0 mg CYC/mL. CYC increased NADP by a similar extent in B and C lung slices. In B slices, NAD losses were approximately 4 times the increases in NADP. CYC did not decrease ATP in B slices and ATP dropped 25% only after 4 hr in the resistant C slices. We conclude that CYC is directly toxic to lung tissue and observe that strain sensitivity in vitro mirrors the sensitivity to fibrosis in vivo. PAP activation and oxidate stress may contribute to this toxicity.

NAD depletion after in vitro exposure of murine lung slices to bleomycin

Bleomycin (BLM), a DNA-cleaving, antitumor antibiotic, causes pulmonary fibrosis. It also causes cell injury and activates the nuclear enzyme poly(ADP-ribose) polymerase (PAP; EC 2.4.2.30) in lung slices exposed to the drug in vitro. 3-Aminobenzamide (3-AB), aPAP inhibitor, prevents enzyme activation and cell injury. We have examined the potential role of ATP and NAD depletion in injury of BLM-sensitive C57B1/6N and -resistant BALB/cN murine lung slices treated with BLM or deprived of glucose, the major metabolic substrate of lung. Lung slices either were treated for 45 min with injurious concentrations of BLM (10–500 μg/mL) or were incubated without glucose, in the presence or absence of 2.5 mM 3-AB. Only the highest concentration of BLM, 500 μg/mL, caused any ATP depletion, and this 35% decrease was transient, occurring at 220 min in C57B1/6N slices. In contrast, glucose deprivation caused 50–70% ATP depletion in slices from both strains. BLM alone at 100 and 500 μg/mL caused a sustained 30–70% NAD depletion from 75 min through 400 min in C57B1/6N mouse lung slices. In the resistant BALB/cN lung slices, NAD depletion by BLM was only seen at 400 min. 3-AB almost completely antagonized NAD depletion in slices from both strains. In contrast to BLM, glucose deprivation did not decrease NAD levels unless 3-AB was present in C57B1/6N slices. Thus, ATP depletion may play a role in the injurious effects of glucose deprivation, but does not appear to be a major factor in pneumocyte injury caused by BLM. NAD depletion or other effects of PAP activation appear to account for the strain-selective, injurious effect of BLM on lung tissue.