American Journal of Respiratory Cell and Molecular Biology

Barnes, Frances A.; Bingle, Lynne; Bingle, Colin D.

doi: 10.1165/rcmb.2007-0388trpmid: 17975173

Murphy, Jaime; Summer, Ross; Wilson, Andrew A.; Kotton, Darrell N.; Fine, Alan

doi: 10.1165/rcmb.2007-0224rcpmid: 18192503

To further examine the half-life of alveolar macrophages, chimeric CD 45.2 mice were generated through bone marrow transplantation of donor CD 45.1 cells. Before administration of donor cells, recipient mice were divided into two cohorts: the first cohort received total body irradiation; the second cohort also received irradiation—however, the thorax, head, and upper extremities were shielded with lead. Flow cytometric analysis was then performed on blood, peritoneal, and bronchoalveolar lavage cells over time to quantify engraftment. The data generated for the unshielded cohort of mice revealed a macrophage half-life of 30 days. In the shielded cohort, however, we found that by 8 months there was negligible replacement of recipient alveolar macrophages by donor cells, despite reconstitution of the blood and peritoneum by donor bone marrow. Consistent with these findings, the mean fluorescent intensity of alveolar macrophages remained stable over a 4-week period after in vivo PKH26 dye loading. Together, these data show that previous alveolar macrophage half-life studies were confounded by the fact that they did not account for the toxic effects of irradiation conditioning regimens, and demonstrate that the bone marrow does not significantly contribute to the alveolar macrophage compartment during steady-state conditions.

Jian, Ming-Yuan; King, Judy A.; Al-Mehdi, Abu-Bakr; Liedtke, Wolfgang; Townsley, Mary I.

doi: 10.1165/rcmb.2007-0192ocpmid: 17962608

High vascular pressure targets the lung septal network, causing acute lung injury. While calcium entry in septal endothelium has been implicated, the channel involved is not known. This study tested the hypothesis that the vanilloid transient receptor potential channel, TRPV4, is a critical participant in the permeability response to high vascular pressure. Isolated lungs from TRPV4+/+ or TRPV4−/− mice were studied at baseline or during high pressure challenge. Permeability was assessed via the filtration coefficient. Endothelial calcium transients were assessed using epifluorescence microscopy of the lung subpleural network. Light microscopy and point counting were used to determine the alveolar fluid volume fraction, a measure of alveolar flooding. Baseline permeability, calcium intensity, and alveolar flooding were no different in TRPV4+/+ versus TRPV4−/− lungs. In TRPV4+/+ lungs, the high pressure–induced permeability response was significantly attenuated by low calcium perfusate, the TRPV antagonist ruthenium red, the phospholipase A2 inhibitor methyl arachidonyl fluorophosphonate, or the P450 epoxygenase inhibitor propargyloxyphenyl hexanoic acid. Similarly, the high pressure–induced calcium transient in TRPV4+/+ lungs was attenuated with ruthenium red or the epoxygenase inhibitor. High vascular pressure increased the alveolar fluid volume fraction compared with control. In lungs from TRPV4−/− mice, permeability, calcium intensity, and alveolar fluid volume fraction were not increased. These data support a role for P450-derived epoxyeicosatrienoic acid–dependent regulation of calcium entry via TRPV4 in the permeability response to high vascular pressure.

McGrath-Morrow, Sharon; Rangasamy, Tirumalai; Cho, Cecilia; Sussan, Thomas; Neptune, Enid; Wise, Robert; Tuder, Rubin M.; Biswal, Shyam

doi: 10.1165/rcmb.2007-0104ocpmid: 17975176

In infants, smoke exposure is associated with more respiratory illnesses and decreased lung function. We hypothesized that perinatal lung is particularly susceptible to the damaging effects of cigarette smoke (CS) and that exposure to CS during this period may alter expression of immune response genes and adversely affect lung growth. To test this, we exposed neonatal mice to 14 days of CS. Immediately after exposure to CS, pulmonary gene expression profiling was performed on 2-week-old CS-exposed lung and age-matched control lung. Nitrotyrosine, TUNEL, MAC3, and phospho-SMAD-2 (p-SMAD2) staining was also performed. At 8 weeks of age, lung volume measurements were determined and mean linear intercept measurements were calculated. Pulmonary gene expression profiling revealed that CS exposure significantly inhibited type 1 and type 2 interferon pathway genes in neonatal lung, compared with age-matched control lung. Neonatal CS-exposed lung also had a significant increase in n-tyrosine, TUNEL, and p-SMAD2 staining when compared with adult CS-exposed lung and age-matched control lung. Lung volumes at 8 weeks of age were modestly but significantly decreased in mice exposed to CS in the neonatal period compared with age-matched controls, consistent with impaired lung growth. The results of this study indicate that exposure to CS during the neonatal period inhibits expression of genes involved in innate immunity and mildly impairs postnatal lung growth. These findings may in part explain the increased incidence of respiratory symptoms in infants and children exposed to CS.

Rhein, Lawrence M.; Perkins, Michael; Gerard, Norma P.; Gerard, Craig

doi: 10.1165/rcmb.2007-0309ocpmid: 17975174

Defenses against bacterial infections involve activation of multiple systems of innate immunity, including complement, Toll-like receptors, and defensins. Reactions to chronic infections bring adaptive immune mechanisms into play as well, with the introduction of modulatory interactions between the two. In humans with chronic lung infections, the severity of inflammation and disease correlate with elevated levels of pathogen-specific immune complexes and complement activation. In mice with genetic deficiency in C5, or targeted deletion of the C5a receptor, Pseudomonas lung infections reveal a role for the C5a anaphylatoxin in disease severity. Deficient animals exhibit significantly reduced survival and clearance of infecting bacteria, simultaneous with greatly increased pulmonary influx of inflammatory cells. Among the actions of C5a on inflammatory cells mediated through the C5a receptor is a shift in the relative expression of Fcγ receptors to increase FcγRIII relative to FcγRII. This shift may significantly impact defenses against chronic infection, reflecting the cellular activation profiles of these IgG receptors. We addressed the role of FcγRIII in defense against Pseudomonas lung infection, and found that, like C5aR-deficient mice, animals with targeted deletion of FcγRIII are more susceptible to mortality upon infection and exhibit reduced clearance of the pathogen. Pseudomonas infection was associated with an increase in the FcγRIII/FcγRII ratio in wild-type mice, and the data support its role as an additional mechanism of host defense against bacterial infection.

Ito, Satoru; Kume, Hiroaki; Naruse, Keiji; Kondo, Masashi; Takeda, Naoya; Iwata, Susumu; Hasegawa, Yoshinori; Sokabe, Masahiro

doi: 10.1165/rcmb.2007-0259ocpmid: 17975175

In response to mechanical stretch, airway smooth muscle exhibits various cellular functions such as contraction, proliferation, and cytoskeletal remodeling, all of which are implicated in the pathophysiology of asthma. We tested the hypothesis that mechanical stretch of airway smooth muscle cells increases intracellular Ca2+ concentration ([Ca2+]i) by activating stretch-activated (SA) nonselective cation channels. A single uniaxial stretch (3 s) was given to human bronchial smooth muscle cells cultured on an elastic silicone membrane. After the mechanical stretch, a transient increase in [Ca2+]i was observed. The [Ca2+]i increase was significantly dependent on stretch amplitude. The augmented [Ca2+]i due to stretch was completely abolished by removal of extracellular Ca2+ and was markedly attenuated by an application of Gd3+, an inhibitor of SA channels, or ruthenium red, a transient receptor potential vanilloid (TRPV) inhibitor. In contrast, the stretch-induced rises of [Ca2+]i were not altered by other Ca2+ channel inhibitors such as nifedipine, BTP-2, and SKF-96365. Moreover, the [Ca2+]i increases were not affected by indomethacin, a cyclooxygenase inhibitor, U-73122, a phospholipase C inhibitor, or xestospongin C, an inhibitor of the inositol-trisphosphate receptor. These findings demonstrate that a novel Ca2+ influx pathway activated by mechanical stretch, possibly through the Ca2+-permeable SA channel activated directly by stretch rather than by indirect mechanisms via intracellular messenger production, is involved in human airway smooth muscle cells. A molecular candidate for the putative SA channel may be one of the members of the TRPV channel family. Thus, abnormal Ca2+ homeostasis in response to excessive mechanical strain would contribute to the pathogenesis of asthma.

Yang, Jing-Qing; Rüdiger, Jochen J.; Hughes, J. Margaret; Goulet, Stephanie; Gencay-Cornelson, Mesut M.; Borger, Pieter; Tamm, Michael; Roth, Michael

doi: 10.1165/rcmb.2007-0079ocpmid: 17989362

The glucocorticoid receptor (GR) is a major control factor for proliferation, differentiation, and inflammation. Our knowledge about the GR is focused on its function as a transcription regulator. However, cells do not always respond to steroids in the same way or develop resistance. The mechanism underlying such a modified steroid response is not well understood, and may depend on the microenvironment of the cells or on the stage of their differentiation. Therefore, we studied the effect of cell density and inflammatory conditions on the expression, compartmentalization, activation, and the anti-proliferative function of the GR in primary human lung fibroblast cultures. In subconfluent cells the GR was located perinuclear, while in confluent cells it was ubiquitously expressed. Serum stimulation up-regulated the level of GR mRNA and protein under all conditions. In subconfluent cells dexamethasone activated the nuclear accumulation and DNA binding of the GR persistently, while in confluent cells its activity declined after 6 hours. In subconfluent cells, but not in confluent cells, the GR interacted with a 42-kD, but not the 30-kD C/EBP-α isoprotein, which resulted in an up-regulation of p21(Waf1/Cip1) expression and suppression of proliferation. In confluent cells, glucocorticoids induced p27(Kip1) expression via p38 mitogen-activated protein kinase and a 52-kD C/EBP-β isoprotein. However, p27(Kip1) did not mediate the antiproliferative effect of glucocorticoids, but simultaneous inhibition of p21(Waf1/Cip1) and p27(Kip1) unlocked contact inhibition in confluent cells. Our results indicate that cell density and serum exposure alter the localization and function of the GR.

Livraghi, Alessandra; Mall, Marcus; Paradiso, Anthony M.; Boucher, Richard C.; Ribeiro, Carla M. Pedrosa

doi: 10.1165/rcmb.2007-0177ocpmid: 17989361

In cystic fibrosis (CF), the absence of functional CFTR leads to dysregulated Na+ absorption across airway epithelia. We established an in vitro model of dysregulated Na+ absorption by treating polarized normal human bronchial epithelial cells (HBEs) with nystatin (Nys), a polyene antibiotic that enables monovalent cations to permeate biological membranes. Acute mucosal Nys produced a rapid increase in short circuit current (Isc) that reflected increased transepithelial Na+ absorption and required Na+/K+ATPase activity. The acute increase in Isc was associated with increased mucosal liquid absorption. Prolonged mucosal Nys treatment resulted in sustained Na+ hyperabsorption, associated with increased mucosal liquid absorption in comparison with naïve (nontreated, kept under air–liquid interface conditions) or vehicle-treated cultures. Nys treatment was not toxic. Increased lactate accumulation in Nys-treated culture media suggested a higher metabolic rate associated with the higher energy demand for Na+ transport. After chronic Nys treatment, the increased Isc was rapidly lost when the cultures were mounted in Ussing chambers, indicating that Nys could be rapidly removed from the apical membrane. Importantly, chronic Nys treatment promoted sustained mucosal liquid depletion and caused mucus dehydration, compaction, and adhesion to the apical surface of Nys-treated cultures. We conclude that mucosal Nys treatment of HBEs provides a simple in vitro model to recapitulate the Na+ and volume hyperabsorptive features of CF airway epithelia.

McGowan, Stephen E.; Holmes, Amey J.; Mecham, Robert P.; Ritty, Timothy M.

doi: 10.1165/rcmb.2007-0281ocpmid: 18006876

Development of the extracellular matrix is a critical feature of alveolar formation and actively involves pulmonary interstitial fibroblasts. The elastic fiber network is an interconnected system of load-bearing fibers that also influences the behavior of adjacent cells, particularly the interstitial lung fibroblasts (LF). We hypothesized that discrete domains of fibrillins-1 and -2 interact with LF integrins and direct their migration in the presence of platelet-derived growth factor (PDGF)-A. Surfaces coated with recombinant peptides lacking or including an arginine-glycine-aspartic acid (RGD) motif were used to study LF migration across porous filters and on protein-coated glass. Exon 24 of fibrillin-2 (Fib2 24), which encodes for an RGD-containing transforming growth factor-β–binding (TB) domain, stimulated migration with greater directional persistence and more effectively stimulated trans-filter migration at low concentrations. Exons 36–44 of fibrillin-1 (Fib1 36–44), which include epidermal growth factor–like domains and an RGD-containing TB domain, induce more lamlellipodia and more widespread remodeling of the leading edge, resulting in greater migration velocity than did Fib2 24. Distinct structural features in regions that surround the RGD motifs may differentially regulate how the PDGF receptor-α promotes integrin distribution and actin filament remodeling at the cell's leading edge. Understanding how fibrillins regulate LF migration may help elucidate how the elastic fiber system could be restored as an interconnected unit, which fails to occur in emphysematous lungs.

Deshmukh, Hitesh S.; Shaver, Colleen; Case, Lisa M.; Dietsch, Maggie; Wesselkamper, Scott C.; Hardie, William D.; Korfhagen, Thomas R.; Corradi, Massimo; Nadel, Jay A.; Borchers, Michael T.; Leikauf, George D.

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