Exaggerated Neurogenic Inflammation and Substance P Receptor Upregulation in RSV-Infected Weanling RatsKing, Katherine A.; Hu, Chengping; Rodriguez, Maria M.; Romaguera, Rita; Jiang, Xiaobo; Piedimonte, Giovanni
doi: 10.1165/ajrcmb.24.2.4264pmid: 11159042
Respiratory syncytial virus (RSV) infection in adult rats causes exaggerated inflammation after sensory nerve stimulation in the extrapulmonary, but not in the intrapulmonary airways. The goal of this study was to analyze neurogenic inflammation in weanling F-344 rats infected with RSV 18 ± 2 d after birth. Five days after RSV inoculation, the extravasation of Evans blue-labeled albumin after nerve stimulation was significantly greater in the intrapulmonary airways of RSV-infected weanling rats than in pathogen-free control rats. In contrast, no difference was found in the extrapulmonary airways. The level of messenger RNA (mRNA) encoding the substance P (SP) receptor (neurokinin 1 [NK1]) increased fourfold in RSV-infected lungs, whereas mRNA encoding the VIPR1 receptor for the antiinflammatory vasoactive intestinal peptide (VIP) increased to a much lesser degree. mRNAs encoding the other neurokinin (NK2) and VIP (VIPR2) receptors were not affected by the virus. Selective inhibition of the NK1 receptor abolished neurogenic inflammation in RSV-infected intrapulmonary airways. Also, neurogenic inflammation and NK1 receptor upregulation in infected lungs were inhibited by prophylaxis with a monoclonal antibody against RSV. These data suggest that RSV lower respiratory tract infection makes the intrapulmonary airways of young rats abnormally susceptible to the proinflammatory effects of SP by selectively upregulating the expression of NK1 receptors.
Endocytosis of Ultrafine Particles by A549 CellsStearns, Rebecca C.; Paulauskis, Joseph D.; Godleski, John J.
doi: 10.1165/ajrcmb.24.2.4081pmid: 11159043
Alveolar epithelium's capacity to ingest inhaled ultrafine particles is not well characterized. The objectives of this study were to use an in vitro model of type II lung epithelium and evaluate the cells' ability to take up ultrafine particles (titanium dioxide [TiO2], 50 nm diameter). The human epithelial cell line A549 was grown on aclar substrates and exposed to 40 μ g/ml TiO2 particles for 3, 6, and 24 h before imaging with energy-filtering transmission electron microscopy. Elemental mapping and electron energy loss spectroscopy were used to colocalize Ti/O with electron-dense particles. Particle endocytosis was compared in A549 cells with and without pretreatment with cytochalasin D (cyto D) (2 μ g/ml). After 3 h of TiO2 exposure, cells internalized aggregates of the ultrafine particles which were observed in cytosolic, membrane-bound vacuoles. After 24 h of exposure there were considerably more intracellular aggregates of membrane-bound particles, and aggregated particles were also enmeshed in loosely and tightly packed lamellar bodies. Throughout 24 h of exposure a preponderance of particles remained associated with the free surface of the cells and were not internalized. The majority of membrane-bound vacuoles contained aggregates of particles and only occasionally did they contain as few as two or three particles, despite the use of several different approaches to assure the possibility for individual particles to be ingested and detected. There was morphologic evidence of microfilament disturbance, but no evidence of a decrease in internalized particles in cells pretreated with cyto D. Thus, this model of type II epithelium is able to internalize aggregates of ultrafine particles.
In Vitro and In Vivo Inhibition of Interleukin (IL)-5–Mediated Eosinopoiesis by Murine IL-5R α Antisense OligonucleotideLach-Trifilieff, Estelle; McKay, Robert A.; Monia, Brett P.; Karras, James G.; Walker, Christoph
doi: 10.1165/ajrcmb.24.2.4237pmid: 11159044
The unique role of interleukin (IL)-5 in eosinophil production, activation, and localization makes this cytokine a prime target for therapeutic intervention in diseases characterized by a selective blood and tissue eosinophilia. In an attempt to block the effects of IL-5 on eosinophils, a strategy was developed to suppress the expression of the IL-5 receptor α chain (IL-5R α ) by antisense oligonucleotides (ASOs). IL-5R α ASOs were identified which selectively and specifically suppress the expression of messenger RNA and proteins of both the membrane and the soluble form of the receptor in constitutively IL-5R–expressing murine BCL-1 cells in vitro. Moreover, these IL-5R α –specific ASOs were able to selectively inhibit the IL-5–induced eosinopoesis from murine fetal liver and bone marrow cells in vitro, suggesting that these molecules may affect the development of IL-5–mediated eosinophilia in vivo. Indeed, intravenous administration of IL-5R α –specific ASOs not only suppressed the bone-marrow and blood eosinophilia in mice after short-term treatment with recombinant murine IL-5 but also inhibited the development of blood and tissue eosinophilia in a ragweed-induced allergic peritonitis model. Thus, blocking the expression of IL-5R α on eosinophil using ASOs may have therapeutic benefits in eosinophilic diseases such as asthma.
Cytokine-Induced Bronchoconstriction in Precision-Cut Lung Slices Is Dependent upon Cyclooxygenase-2 and Thromboxane Receptor ActivationMartin, Christian; Uhlig, Stefan; Ullrich, Volker
doi: 10.1165/ajrcmb.24.2.3545pmid: 11159047
Cytokines play an essential role in the regulation of inflammatory responses. The effects of cytokines on lung functions are less well known and their study in vivo is complicated by the attraction of leukocytes to the inflamed sites. Recently the model of precision-cut lung slices was developed, where viable lung slices with an intact microanatomy are taken into culture and where bronchoconstriction can be followed by observing single airways under the microscope. We used this model to study the direct effects of cytokines on airway tonus in the absence of blood-derived leukocytes. Incubation of precision-cut lung slices with a mixture of tumor necrosis factor (TNF)-α, interleukin (IL)-1 β, and interferon (IFN)-γ resulted in contraction of airways, which was accompanied by expression of cyclooxygenase (Cox)-2 and thromboxane release into the supernatant. The thromboxane receptor antagonist SQ29548 completely prevented the cytokine-induced bronchoconstriction, whereas the 5-lipoxygenase inhibitor AA681 had no effect on cytokine-induced bronchoconstriction. Preventing the expression of Cox-2 by dexamethasone or blocking Cox-2 activity with the selective Cox-2 inhibitor NS398 attenuated both thromboxane formation and bronchoconstriction. Incubation of lung slices with each of the cytokines alone caused no bronchoconstriction; in fact, IL-1 alone rather dilated the airways. However, simultaneous incubation with TNF and IL-1 β caused a bronchoconstriction that was not further enhanced by IFN-γ. We conclude that TNF-α and IL-1 β synergistically cause bronchoconstriction by induction of Cox-2 and subsequent activation of the thromboxane receptor. Our study raises the possibility that TNF and IL-1 may contribute to bronchospasm during inflammatory lung diseases.
Mast Cell Tryptase Activates Extracellular-Regulated Kinases (p44/p42) in Airway Smooth-Muscle Cells: Importance of Proteolytic Events, Time Course, and Role in Mediating MitogenesisBrown, James K.; Jones, Cary A.; Rooney, LeeAnn A.; Caughey, George H.
doi: 10.1165/ajrcmb.24.2.4165pmid: 11159048
We previously reported that mast cell tryptase is a potent mitogen for cultured airway smooth-muscle cells, but the early intracellular signals mediating this response are not known. In many cells, proliferative effects are mediated by a mitogen-activated protein kinase signaling pathway involving Raf-1, MAP kinase kinases (MEKs), and extracellular signal-regulated protein kinases (ERKs) 1 and 2. Therefore, we tested for tryptase-induced activation of ERK1 and 2 in cultured dog tracheal smooth-muscle cells. Tryptase, in nanomolar concentrations which potently stimulated DNA synthesis, increased dual phosphorylation of ERKs in cellular lysates as well as ERK2 kinase activity in immunoprecipitates. Pretreatment of cells with the MEK inhibitor PD098059 abolished tryptase-induced increases in DNA synthesis and attenuated increases in ERK2 activity. Irreversible inhibition of tryptase's proteolytic activity, using p-amidino phenylmethanesulfonyl fluoride, attenuated tryptase-induced increases in DNA synthesis and dual phosphorylation of ERKs by 76% and 40 to 60%, respectively. Tryptase also increased c-fos transcription as quantified in polymerase chain reactions. In concentrations that caused similar increases in DNA synthesis, tryptase and platelet-derived growth factor (PDGF-BB) increased ERK activity (and c-fos transcription) with markedly different kinetics, the tryptase-induced responses being slower in onset and more sustained. We conclude that tryptase-induced mitogenesis in airway smooth-muscle cells requires activation of ERK1 and 2; that these responses depend partially, but not completely, upon tryptase's properties as a protease; and that they are slower in onset and more sustained than those induced by PDGF-BB.
Regulation of G Protein–Coupled Receptor–Adenylyl Cyclase Responsiveness in Human Airway Smooth Muscle by Exogenous and Autocrine AdenosineMundell, Stuart J.; Olah, Mark E.; Panettieri, Reynold A.; Benovic, Jeffrey L.; Penn, Raymond B.
doi: 10.1165/ajrcmb.24.2.4243pmid: 11159049
Adenosine is a mediator of bronchoconstriction in asthmatics and is believed to mediate its effects through adenosine receptor activation in inflammatory cells. In this study, we identify human airway smooth muscle (ASM) as a direct target of adenosine. Acute exposure of human ASM cultures to adenosine receptor (AR) agonists resulted in rapid accumulation of cyclic adenosine monophosphate (cAMP) with a pharmacologic profile consistent with A2bAR activation. Little or no evidence of A1AR or A3AR expression was suggested on acute addition of various AR ligands, although a low level of A1ARs was identified in radioligand binding studies. Treatment with adenosine deaminase suggested that human ASM cultures secrete adenosine that feeds back on A2bARs and regulates basal cAMP levels as well as a small degree of A2bAR, β2AR, and prostaglandin E2 receptor desensitization. When subjected to chronic treatment with AR agonists or agents that enhance accumulation of endogenous, extracellular adenosine, a dual effect of A2bAR desensitization and adenylyl cyclase (AC) sensitization was observed. This AC sensitization was eliminated by pertussis toxin and partially reversed by the A1AR antagonist 8-cyclopentyl-1,3-dipropylxanthine, suggesting a contributory role for the A1AR. Overexpression of A1ARs and A2bARs in human ASM cultures resulted in differential effects on basal, agonist-, and AC-mediated cAMP production. These data demonstrate that human ASM is a direct target of exogenous and autocrine adenosine, with effects determined by differential contributions of A2b and A1 adenosine receptors that are time-dependent. Accordingly, the relative distribution and activation of AR subtypes in ASM in vivo may influence airway function in diseases such as asthma and warrant consideration in therapeutic strategies that target ARs or alter nucleotide/nucleoside levels in the airway.
Differential Induction of TNF-α and MnSOD by Endotoxin: Role of Reactive Oxygen Species and NADPH OxidaseWhite, Julie E.; Tsan, Min-Fu
doi: 10.1165/ajrcmb.24.2.4169pmid: 11159050
Endotoxin (lipopolysaccharide [LPS]) is known to induce the production of tumor necrosis factor (TNF)-α and the induction of manganese superoxide dismutase (MnSOD). We have recently demonstrated that induction of TNF-α and MnSOD by LPS is mediated through different signal transduction pathways. In the current study, we investigated the role of reactive oxygen species (ROS) in the induction of TNF-α and MnSOD messenger RNAs (mRNAs) in human monocytes. Hypoxia (1% O2) inhibited the production of superoxide (O2−) and the induction of MnSOD, but not TNF-α, mRNA. Diphenylene iodonium (DPI), a potent inhibitor of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, had no effect on LPS induction of MnSOD mRNA, whereas it markedly inhibited LPS-induced O2− production. Neither hypoxia nor DPI had any effect on LPS activation of nuclear factor (NF)-κ B. These results suggest that (1) ROS is important in the induction of MnSOD, but not TNF-α, mRNA by LPS, (2) ROS from sources other than NADPH oxidase is involved in LPS induction of MnSOD mRNA, and (3) ROS-mediated LPS induction of MnSOD mRNA is independent of NF-κ B activation.