Treatment Of Airway Inflammatory Diseases Research Articles

Strategic Formulation Study on Dry Powder Inhalation System Based on Modulated Molecular Properties and Controlled Pharmacokinetics

Cyclosporine A (CsA) has been widely used as an immunosuppressive agent, and recent outcomes from clinical studies are indicative of the potent therapeutic potential of CsA for chronic asthma and airway inflammation. The clinical use of CsA for airway inflammatory diseases is partly limited because of low oral bioavailability and severe systemic side effects. A number of CsA dosage forms have been proposed to overcome these drawbacks, for example, nebulizer formulation and metered-dose inhaler formulation for inhalation therapy, whereas these liquid formulations sometimes contain organic solvents and other solubilizers, leading to local irritant potency. In this context, our group developed a dry powder inhalation (DPI) system of CsA, employing a polymer-based amorphous solid dispersion (ASD) approach, for inhalation therapy on airway inflammations. There was marked improvement in dissolution behavior of the ASD formulation compared with that of an amorphous CsA. The new DPI system of CsA exhibited high dispersibility and suitable particle distribution for inhalation therapy. In vivo experiments demonstrated that inhaled DPI system of CsA attenuated inflammatory events in experimental asthma/chronic obstructive pulmonary disease (COPD) model rats as evidenced by a decrease of infiltrated granulocytes, and there was no excessive increase in systemic exposure of CsA at a pharmacologically effective dose, possibly leading to reduced systemic side effects. From these findings, combination use of CsA-loaded ASD and DPI systems might be a promising approach for the treatment of airway inflammatory diseases with improved pharmacodynamics and lower systemic exposure.

Effect of Anthocyanidin on MUC5AC and MUC5B Expression in Airway Epithelial Cells

Received March 11, 2013 Revised April 18, 2013 Accepted April 21, 2013 Address for correspondence Yong-Dae Kim, MD, PhD Department of OtorhinolaryngologyHead and Neck Surgery, College of Medicine, Yeungnam University, 170 Hyeonchung-ro, Nam-gu, Daegu 705-703, Korea Tel +82-53-620-3781 Fax +82-53-628-7884 E-mail ydkim@med.yu.ac.kr Background and ObjectivesZZNaringenin and delphinidin are types of anthocyanidin, which are flavonoids and thus have anti-inflammatory property. Moderate consumption of natural dietary naringenin and delphinidin is believed to do anti-inflammatory action, but the action mechanism is unclear. Therefore, this study aimed to investigate the effects of naringenin and delphinidin on interleukin-1β (IL-1β)and lipopolysaccharide (LPS)-induced MUC5AC and MUC5B expressions in airway epithelial cells. Materials and MethodZZIn NCI-H292 cells and cultured nasal polyp epithelial cells, the effects of naringenin and delphinidin on IL-1βand LPS-induced MUC5AC and MUC5B expressions were analyzed by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. ResultsZZDelphinidin attenuated IL-1βand LPS-induced MUC5AC and MUC5B mRNA and glycoprotein expression in a dose-dependent pattern in NCI-H292 cells and in cultured nasal polyp epithelial cells. Naringenin partially attenuated IL-1βand LPS-induced MUC5AC and MUC5B mRNA and glycoprotein expression at a high dose. ConclusionZZThese results suggest that delphinidin attenuates MUC5AC and MUC5B expressions in the airway epithelial cells. Between anthocyanidin and delphinidin, delphinidin exhibits greater potential as an ideal therapeutic agent for the control of mucus-hypersecretion in the treatment of airway inflammatory diseases. Korean J Otorhinolaryngol-Head Neck Surg 2013;56:291-6

Inhalable sustained-release formulation of long-acting vasoactive intestinal peptide derivative alleviates acute airway inflammation

The present study was undertaken to develop a respirable sustained-release powder (RP) formulation of long-acting VIP derivative, [Arg15, 20, 21, Leu17]-VIP-GRR (IK312532), using PLGA nanospheres (NS) with the aim of improving the duration of action. NS formulation of IK312532 (IK312532/NS) was prepared by an emulsion solvent diffusion method in oil, and a mixture of the IK312532/NS and erythritol was jet-milled and mixed with lactose carrier to obtain the IK312532/NS-RP. Physicochemical properties were characterized focusing on appearance, particle size, and drug release, and in vivo pharmacological effects were assessed in antigen-sensitized rats. The IK312532/NS with a diameter of 140nm showed a biphasic release pattern in distilled water with ca. 20% initial burst for 30min and a sustained slow release up to ca. 55% for 24h. Laser diffraction analysis demonstrated that IK312532/NS-RP had fine dispersibility and suitable particle size for inhalation. In antigen-sensitized rats, insufflated IK312532/NS-RP (10μg of IK312532/rat) could suppress increases of granulocyte recruitment and myeloperoxidase in pulmonary tissue for up to 24h after antigen challenge, although IK312532-RP at the same dose was less effective with limited duration of action. From these findings, newly prepared IK312532/NS-RP might be of clinical importance in improving duration of action and medication compliance for treatment of airway inflammatory diseases.

Inhalable dry-emulsion formulation of cyclosporine A with improved anti-inflammatory effects in experimental asthma/COPD-model rats

The main purpose of the present study was to develop a novel respirable powder (RP) formulation of cyclosporine A (CsA) using a spray-dried O/W-emulsion (DE) system. DE formulation of CsA (DE/CsA) was prepared by spray-drying a mixture of erythritol and liquid O/W emulsion containing CsA, polyvinylpyrrolidone, and glyceryl monooleate as emulsifying agent. The DE/CsA powders were mixed with lactose carriers to obtain an RP formulation of DE/CsA (DE/CsA-RP), and its physicochemical, pharmacological, and pharmacokinetic properties were evaluated. Spray-dried DE/CsA exhibited significant improvement in dissolution behavior with ca. 4500-fold increase of dissolution rate, and then, nanoemulsified particles were reconstituted with a mean diameter of 317 nm. Laser diffraction analysis on the DE/CsA-RP suggested high dispersion of DE/CsA on the surface of the lactose carrier. Anti-inflammatory properties of the inhaled DE/CsA-RP were characterized in antigen-sensitized asthma/COPD-model rats, in which the DE/CsA-RP was more potent than the RP formulation of physical mixture containing CsA and erythritol in inhibiting inflammatory responses, possibly due to the improved dissolution behavior. Pharmacokinetic studies demonstrated that systemic exposure of CsA after intratracheal administration of the DE/CsA-RP at a pharmacologically effective dose (100 μg-CsA/rat) was 50-fold less than that of the oral CsA dosage form at a toxic dose (10 mg/kg). From these findings, use of inhalable DE formulation of CsA might be a promising approach for the treatment of airway inflammatory diseases with improved pharmacodynamics and lower systemic exposure.

Anti-inflammatory effects of garenoxacin on IL-8 production and ERK1/2 activation induced by lipopolysaccharides in A549 and THP-1 cells

The anti-inflammatory properties of macrolides have been applied to the treatment of inflammatory airway diseases. Although the anti-inflammatory properties of fluoroquinolones have been reported, no reports are available regarding a newly developed fluoroquinolone, garenoxacin (GRNX). To examine the immunomodulatory effect of GRNX, we examined the transcription and secretion of inflammatory cytokines by human airway epithelial cells and monocytes stimulated with lipopolysaccharide (LPS). A human lung epithelial cell line (A549) and a human monocyte cell line (THP-1) were stimulated with LPS and exposed to different concentrations of GRNX. The transcription and secretion of interleukin 8 (IL-8) in both A549 and THP-1 cells was measured by real-time PCR and an enzyme-linked immunosorbent assay, respectively. Treatment with GRNX significantly inhibited the transcription and secretion of IL-8 induced by LPS-stimulated cells through inhibitory ERK1/2 phosphorylation. GRNX has anti-inflammatory activity through its capacity to alter the secretion of IL-8 from A549 and THP-1 cell lines. Our findings suggest that GRNX is suitable for the treatment of LPS-induced respiratory infection and inflammatory airway diseases.

The anti-inflammatory activities of an extract and compounds isolated from Platycladus orientalis (Linnaeus) Franco in vitro and ex vivo

Ethnopharmacological relevanceAs a Chinese traditional herbal medicine, leaves of Platycladus orientalis (Linnaeus) Franco (LPO) are used to treat coughs, excessive mucus secretion, chronic bronchitis, bronchiectasis, and asthma, etc. The experiments were carried out to investigate their anti-inflammatory properties and mechanisms, which could support the Chinese traditional uses of treating inflammatory airway diseases. Materials and methodsThe anti-inflammatory activities of the chloroform fraction (CHL) and pure compounds of LPO were evaluated for their abilities to inhibit pro-inflammatory enzymes in vitro, and production of tumor necrosis factor-α (TNF-α) and nitric oxide in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Furthermore, the arachidonic acid metabolites, stimulated by calcium ionophore A23187, were also determined by HPLC. ResultsFor the first time, the assays of eicosanoids in intact cells showed that the CHL, hinokiol, and acacetin had significant inhibitory effects on 5-hydroxy-eicosa-tetra-enoic acid (5-HETE) and leukotriene B4 (LTB4) formations. And cell-free enzyme assays (5-lipoxygenase, leukotriene A4-hydrolase, cyclooxgenase-2) demonstrated the potent inhibitory effects of the CHL, hinokiol and acacetin on 5-lipoxygenase (5-LOX). Then, the inhibitions of the CHL, hinokiol on NO biosynthesis and the inhibitions of the CHL, 8(14),15-pimaradien-3β,18-diol, and hinokiol on TNF-α release were also confirmed in the RAW264.7 murine macrophages. ConclusionThe data indicate that the inhibitory effects of the CHL and its components (hinokiol and acacetin) on 5-LOX contribute to the anti-inflammatory activity of LPO. Moreover, the CHL and its components also show beneficial effects on NO and TNF-α production. Consequently, these results provide a rationale for LPO's traditional applications in the treatment of inflammatory airway diseases.

Platelet membranes induce airway smooth muscle cell proliferation

The role of platelets in airway disease is poorly understood although they have been suggested to influence on proliferation of airway smooth muscle cells (ASMC). Platelets have been found localized in the airways in autopsy material from asthmatic patients and have been implicated in airway remodeling. The aim of the present study was to investigate the effects of various platelet fractions on proliferation of ASMC obtained from guinea pigs (GP-ASMC) and humans (H-ASMC). Proliferation of ASMC was measured by the MTS assay and the results confirmed by measurements of the DNA content. A key observation was that the platelet membrane preparations induced a significant increase in the proliferation of both GP-ASMC (129.9 ± 3.0 %) and H-ASMC (144.8 ± 12.2). However, neither supernatants from lysed or filtrated thrombin stimulated platelets induced ASMC proliferation to the same extent as the membrane preparation. We have previously shown that platelet-induced proliferation is dependent on 5-lipoxygenase (5-LOX) and reactive oxygen species (ROS) pathways. In the present work we established that platelet membrane-induced ASMC proliferation was reduced in the presence of the NADPH oxidase inhibitor DPI and the 5-LOX inhibitor AA-861. In conclusion, our results showed that platelet membranes significantly induced ASMC proliferation, demonstrating that the mitogenic effect of platelets and platelet membranes on ASMC is mainly due to membrane-associated factors. The effects of platelet membranes were evident on both GP-ASMC and H-ASMC and involved 5-LOX and ROS. These new findings are of importance in understanding the mechanisms contributing to airway remodeling and may contribute to the development of new pharmacological tools in the treatment of inflammatory airway diseases.

Stable dry powder inhaler formulation of tranilast attenuated antigen-evoked airway inflammation in rats

Tranilast (TL) has been clinically used for the treatment of airway inflammatory diseases, although the clinical use of TL is limited because of its poor solubility and systemic side effects. To overcome these drawbacks, a novel respirable powder of TL (CSD/TL-RP) for inhalation therapy was developed using nanocrystal solid dispersion of TL (CSD/TL). Stability study on CSD/TL-RP was carried out with a focus on inhalation performance. Even after 6 months of storage at room temperature, there were no significant morphological changes in micronized particles on the surface of carrier particles as compared with that before storage. Cascade impactor analyses on CSD/TL-RP demonstrated high inhalation performance with emitted dose and fine particle fraction (FPF) of ca. 98% and 60%, respectively. Long-term storage of CSD/TL-RP resulted in only a slight decrease in FPF value (ca. 54%). Inhaled CSD/TL-RP could attenuate antigen-induced inflammatory events in rats, as evidenced by marked reduction of granulocytes in bronchoalveolar lavage fluid and inflammatory biomarkers such as eosinophil peroxidase, myeloperoxidase, and lactate dehydrogenase. These findings were consistent with decreased expression levels of mRNAs for nuclear factor-kappa B and cyclooxygenase-2, typical inflammatory mediators. Given these findings, inhalable TL formulation might be an interesting alternative to oral therapy for the treatment of asthma and other airway inflammatory diseases with sufficient dispersing stability.

Allergic Rhinitis and Inflammatory Airway Disease: Interactions within the Unified Airspace

Allergic rhinitis (AR), the most common chronic allergic condition in outpatient medicine, is associated with immense health care costs and socioeconomic consequences. AR's impact may be partly from interacting of respiratory conditions via allergic inflammation. This study was designed to review potential interactive mechanisms of AR and associated conditions and consider the relevance of a bidirectional "unified airway" respiratory inflammation model on diagnosis and treatment of inflammatory airway disease. MEDLINE was searched for pathophysiology and pathophysiological and epidemiologic links between AR and diseases of the sinuses, lungs, middle ear, and nasopharynx. Allergic-related inflammatory responses or neural and systemic processes fostering inflammatory changes distant from initial allergen provocation may link AR and comorbidities. Treating AR may benefit associated respiratory tract comorbidities. Besides improving AR outcomes, treatment inhibiting eosinophil recruitment and migration, normalizing cytokine profiles, and reducing asthma-associated health care use in atopic subjects would likely ameliorate other upper airway diseases such as acute rhinosinusitis, chronic rhinosinusitis (CRS) with nasal polyposis (NP), adenoidal hypertrophy, and otitis media with effusion. Epidemiological concordance of AR with several airway diseases conforms to a bidirectional "unified airway" respiratory inflammation model based on anatomic and histological upper and lower airway connections. Epidemiology and current understanding of inflammatory, humoral, and neural processes make links between AR and disorders including asthma, otitis media, NP, and CRS plausible. Combining AR with associated conditions increases disease burden; worsened associated illness may accompany worsened AR. AR pharmacotherapies include antihistamines, leukotriene antagonists, intranasal corticosteroids, and immunotherapy; treatments attenuating proinflammatory responses may also benefit associated conditions.

Selective Prostacyclin Receptor Agonism Augments Glucocorticoid-Induced Gene Expression in Human Bronchial Epithelial Cells

Prostacyclin receptor (IP-receptor) agonists display anti-inflammatory and antiviral activity in cell-based assays and in preclinical models of asthma and chronic obstructive pulmonary disease. In this study, we have extended these observations by demonstrating that IP-receptor activation also can enhance the ability of glucocorticoids to induce genes with anti-inflammatory activity. BEAS-2B bronchial epithelial cells stably transfected with a glucocorticoid response element (GRE) luciferase reporter were activated in a concentration-dependent manner by the glucocorticoid dexamethasone. An IP-receptor agonist, taprostene, increased cAMP in these cells and augmented luciferase expression at all concentrations of dexamethasone examined. Analysis of the concentration-response relationship that described this effect showed that taprostene increased the magnitude of transcription without affecting the potency of dexamethasone and was, thus, steroid-sparing in this simple system. RO3244794, an IP-receptor antagonist, and oligonucleotides that selectively silenced the IP-receptor gene, PTGIR, abolished these effects of taprostene. Infection of BEAS-2B GRE reporter cells with an adenovirus vector encoding a highly selective inhibitor of cAMP-dependent protein kinase (PKA) also prevented taprostene from enhancing GRE-dependent transcription. In BEAS-2B cells and primary cultures of human airway epithelial cells, taprostene and dexamethasone interacted either additively or cooperatively in the expression of three glucocorticoid-inducible genes (GILZ, MKP-1, and p57(kip2)) that have anti-inflammatory potential. Collectively, these data show that IP-receptor agonists can augment the ability of glucocorticoids to induce anti-inflammatory genes in human airway epithelial cells by activating a cAMP/PKA-dependent mechanism. This observation may have clinical relevance in the treatment of airway inflammatory diseases that are either refractory or respond suboptimally to glucocorticoids.

Prostanoids and the Cough Reflex

Prostanoids such as prostaglandin (PG) D(2), PGE(2), PGF(2alpha), prostacyclin (PGI(2)), and thromboxane (Tx) A(2) act via five classes of receptors named DP, EP, FP, IP, and TP, respectively, and mediate a diverse range of physiological effects. Prostanoids are commonly associated with many diseases as a proinflammatory mediator; however, in the lung, prostanoids, particularly PGE(2), seem to have a protective role. Inhaled PGE(2) has been shown to be anti-inflammatory and a bronchodilator but causes cough. This has hindered the development of prostanoids for the treatment of airway inflammatory diseases. We discuss here the extensive research into the role of prostanoids in the airways and their modulation of the cough reflex.

Oxygen and Nitrogen Reactive Species Are Effectively Scavenged by Eucalyptus globulus Leaf Water Extract

Eucalyptus globulus Labill. (Family Myrtaceae) is a plant of Australian origin, with a reported therapeutic use in airway inflammatory diseases. Considering that reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been implicated in the pathogenesis of airway inflammatory diseases such as asthma and chronic obstructive pulmonary disease, an effective scavenging activity against these reactive species may contribute for the therapeutic effect of this plant. In the present study, a water extract of E. globulus leaves was evaluated for its putative in vitro scavenging effects on ROS (HO(*), O(2)(*-), ROO(*), and H(2)O(2)) and RNS ((*)NO and ONOO(-)) and on 2,2-diphenyl-1-picrylhydrazyl radical (DPPH). Qualitative and quantitative analyses of the extract's phenolic composition were also performed. The Eucalyptus leaf water extract presented a remarkable capacity to scavenge all the reactive species tested, with all the 50% inhibitory concentrations being found at the mug/mL level. Phytochemical analysis showed the presence of polyphenols such as flavonoids (rutin and quercitrin) and phenolic acids (chlorogenic acid and ellagic acid), which may be partially responsible for the observed antioxidant activity. These observations provide further support, beyond the well-known antibacterial and antiviral activities of the Eucalyptus plant, for its reported use in traditional medicine such as in the treatment of airway inflammatory diseases, considering the important role of ROS and RNS in the inflammatory process, although further studies are needed to prove the bioavailability of the antioxidants/antibacterial compounds of the extract as well as the ability of the active compounds to reach specific tissues and to act in them.

Mechanistic Systems Biology of Inflammatory Gene Expression in Airway Smooth Muscle as Tool for Asthma Drug Development

There is compelling evidence that airway smooth muscle cells may function as inflammatory cells in the airway system by producing multiple inflammatory cytokines in response to a large array of external stimuli such as acetylcholine, bradykinin, inflammatory cytokines, and toll-like receptor activators. However, how multiple extracellular stimuli interact in the regulation of inflammatory gene expression in an airway smooth muscle cell remains poorly understood. This review addresses the mechanistic systems biology of inflammatory gene expression in airway smooth muscle by discussing: a) redundancy underlying multiple stimulus-product relations in receptor-mediated inflammatory gene expression, and their regulation by convergent activation of Erk1/2 mitogen-activated protein kinase (MAPK), b) Erk1/2 MAPK-dependent induction of phosphatase expression as a negative feedback mechanism in the robust maintenance of inflammatory gene expression, and c) cyclooxygenase 2-dependent regulation of the differential temporal dynamics of early and late inflammatory gene expression. It is becoming recognized that a single-target approach is unlikely to be effective for the treatment of inflammatory airway diseases because airway inflammation is a result of complex interactions among multiple inflammatory mediators and cells types in the airway system. Understanding the mechanistic systems biology of inflammatory gene expression in airway smooth muscle and other cell types in the airway system may lead to the development of multi-target drug regimens for the treatment of inflammatory airway diseases such as asthma.

Reduced Airway Inflammation and Remodeling in Parallel with Mucin 5AC Protein Expression Decreased by S-Carboxymethylcysteine, a Mucoregulant, in the Airways of Rats Exposed to Sulfur Dioxide

Background: Human obstructive airway diseases are histopathologically characterized by inflammatory cell infiltration, goblet cell hyperplasia, and mucus hypersecretion in airways. We prepared a rat model of airway injury by exposure of sulfur dioxide (SO₂) and then evaluated the effects of S-carboxymethylcysteine (S-CMC), a mucoregulant. Methods: Rats were exposed to SO₂ gas for 44 days and orally given S-CMC at 250 mg/kg, twice daily, from 21 to 44 days of exposure for histopathological and immunohistochemical evaluation. Results: SO₂ exposure induced inflammatory cell infiltration and mucus cell increase in rat airways. S-CMC treatment significantly decreased this inflammatory cell infiltration in proximal and peripheral airways. Morphometrically, SO₂ exposure significantly increased the number of Alcian blue (pH 2.5)- and periodic acid-Schiff (AB/PAS)-positive cells in rat airways (11.8 × 10^–2 cell/nuclear profiles per micrometer basement membrane) compared to normal rat airways (1.6 × 10^–2 cell/nuclear profiles per micrometer basement membrane). S-CMC treatment significantly decreased the number of AB/PAS-positive cells (4.4 × 10^–2 cell/nuclear profiles per micrometer basement membrane, p < 0.01 vs. SO₂-exposed rats). Immunohistochemically, SO₂ exposure increased the expression of mucin 5AC (MUC5AC) protein in the airway epithelium of rats, but S-CMC treatment inhibited the increase. Conclusions: The increased mucus cells and MUC5AC protein expression seem associated with SO₂-induced airway inflammation in rats. The fact that S-CMC suppresses airway inflammation and the increase in mucus cells and MUC5AC protein expression suggests that this mucoregulant may be advantageous in the treatment of inflammatory airway diseases with goblet cell hyperplasia.

Kinase targets and inhibitors for the treatment of airway inflammatory diseases: the next generation of drugs for severe asthma and COPD?

Kinases are believed to play a crucial role in the expression and activation of inflammatory mediators in the airway, in T-cell function, and in airway remodeling. Important pro-inflammatory transcription factors such as activating protein-1 and nuclear factor kappaB, which are activated in airway disease, require kinase activation to switch on inflammatory genes, while other kinases can regulate mRNA half-life. Selective kinase inhibitors have been developed that reduce inflammatory gene expression and some characteristics of disease in animal models. Targeting specific kinases that are overexpressed or overactive in disease should allow for selective treatment of airway inflammatory diseases. Interest in this area has intensified due to the success of the specific Abelson murine leukemia viral oncogene homolog tyrosine kinase inhibitor, imatinib mesylate, in the treatment of chronic myelogenous leukemia. Encouraging data from animal models and primary cells and early phase I and II studies in other diseases suggest that inhibitors of p38 mitogen-activated protein kinase and inhibitor of kappaB kinase-2 may prove to be useful novel therapies in the treatment of severe asthma and chronic obstructive pulmonary disease.

Novel therapies for the treatment of inflammatory airway disease

Novel therapies for the treatment of inflammatory airway disease

Role of p38 MAP kinase in LPS-induced airway inflammation in the rat.

We investigated the effect of the p38 kinase inhibitor SB 203580 on airway inflammation induced by aerosolized lipopolysaccharide (LPS) in male Wistar rats. SB 203580 significantly inhibited (ED(50)=15.8 mg kg(-1)) plasma levels of TNF-alpha in rats challenged with LPS (1.5 mg kg(-1), i.p.). Aerosolized LPS induced a peak in TNF-alpha levels and the initiation of a neutrophilic response in bronchoalveolar lavage (BAL) fluid at the 2 h time point. Furthermore, the 4 h time point was associated with the peak in IL-1beta levels and the initial plateau of neutrophilia observed in the BAL fluid. SB 203580 (100 mg kg(-1)), had no effect on peak TNF-alpha levels or the associated neutrophilia in the BAL. Interestingly, the PDE 4 inhibitor RP 73401 (100 mg kg(-1)) significantly reduced both TNF-alpha levels and neutrophilic inflammation. However, the BAL fluid from rats pre-treated with either compound significantly inhibited TNF-alpha release from cultured human monocytes 18 h after LPS treatment (83.6 and 44.5% inhibition, respectively). Alternatively, SB 203580 (100 mg kg(-1)) produced dose-related inhibition of BAL IL-1beta levels (67.5% inhibition, P<0.01) and BAL neutrophilia (45.9% inhibition, P<0.01) 4 h after LPS challenge. P38 protein was present in lung tissue and the level of expression was not affected by LPS treatment. P38 kinase appears to be involved in the release of IL-1beta and the sustained neutrophilic response in the BAL fluid. This data may suggest a role for p38 inhibitors in the treatment of airway inflammatory diseases in which neutrophilia is a feature of the lung pathology.

Glucocorticoids inhibit lipopolysaccharide-induced up-regulation of arginase in rat alveolar macrophages.

1. As arginase by limiting nitric oxide (NO) synthesis may play a role in airway hyperresponsiveness and glucocorticoids are known to induce the expression of arginase I in hepatic cells, glucocorticoid effects on arginase in alveolar macrophages (AM Phi) were studied. 2. Rat AM Phi were cultured in absence or presence of test substances. Thereafter, nitrite accumulation, arginase activity, and the expression pattern of inducible NO synthase, arginase I and II mRNA (RT - PCR) and proteins (immunoblotting) were determined. 3. Lipopolyssacharides (LPS, 20 h) caused an about 2 fold increase in arginase activity, whereas interferon-gamma (IFN-gamma), like LPS a strong inducer of NO synthesis, had no effect. 4. Dexamethasone decreased arginase activity by about 25% and prevented the LPS-induced increase. Mifepristone (RU-486) as partial glucocorticoid receptor agonist inhibited LPS-induced increase by 45% and antagonized the inhibitory effect of dexamethasone. 5. Two different inhibitors of the NF-kappa B-pathway also prevented LPS-induced increase in arginase activity. 6. Rat AM Phi expressed mRNA and protein of arginase I and II, but arginase I expression was stronger. Arginase I mRNA and protein was not affected by IFN-gamma, but increased by LPS and this effect was prevented by dexamethasone. Both, LPS and IFN-gamma enhanced the levels of arginase II mRNA and protein, effects also inhibited by dexamethasone. As IFN-gamma did not affect total arginase activity, arginase II may represent only a minor fraction of total arginase activity. 7. In rat AM Phi glucocorticoids inhibit LPS-induced up-regulation of arginase activity, an effect which may contribute to the beneficial effects of glucocorticoids in the treatment of inflammatory airway diseases.

Continuous nitric oxide synthesis by inducible nitric oxide synthase in normal human airway epithelium in vivo.

Nitric oxide (NO) is an important mediator of inflammatory responses in the lung and a key regulator of bronchomotor tone. An airway NO synthase (NOS; EC 1.14.13.39) has been proposed as a source of endogenous NO in the lung but has not been clearly defined. Through molecular cloning, we conclusively demonstrate that NO synthesis in normal human airways is due to the continuous expression of the inducible NOS (iNOS) isoform in airway epithelial cells. Although iNOS mRNA expression is abundant in airway epithelial cells, expression is not detected in other pulmonary cell types, indicating that airway epithelial cells are unique in the continuous pattern of iNOS expression in the lung. In situ analysis reveals all airway epithelial cell types express iNOS. However, removal of epithelial cells from the in vivo airway environment leads to rapid loss of iNOS expression, which suggests expression is dependent upon conditions and/or factors present in the airway. Quantitation of NOS activity in epithelial cell lysates indicates nanomolar levels of NO synthesis occur in vivo. Remarkably, the high-level iNOS expression is constant in airway epithelium of normal individuals over time. However, expression is strikingly decreased by inhaled corticosteroids and beta-adrenergic agonists, medications commonly used in treatment of inflammatory airway diseases. Based upon these findings, we propose that respiratory epithelial cells are key inflammatory cells in the airway, functioning in host defense and potentially playing a role in airway inflammation.

Corticosteroids increase secretory leukocyte protease inhibitor transcript levels in airway epithelial cells.

Secretory leukocyte protease inhibitor (SLPI) is the predominant antiprotease of the conducting airways and may play a role in reducing airway inflammation. In this study, the effect of corticosteroids used in the treatment of inflammatory airway disease on SLPI transcript levels was investigated. When human airway epithelial cells (9HTEo-) were treated continuously with 10 nM fluticasone propionate, SLPI transcript levels increased within 12 h, with maximal transcript accumulation occurring at 24-48 h. Several corticosteroids (0.1-1,000 nM) were compared, and the following potency in increasing SLPI transcript levels was observed: fluticasone > triamcinolone > or = dexamethasone > methylprednisolone > hydrocortisone. Fluticasone, the most potent corticosteroid, increased SLPI transcript levels at doses as low as 0.1 nM, whereas hydrocortisone, the least potent corticosteroid, was effective at 100 nM. Fluticasone-induced increases in SLPI transcript levels were inhibited by cycloheximide, suggesting protein synthesis may be required for this response. Because proteases are likely to be present when corticosteroids are administered therapeutically, we examined the interaction between elastase and fluticasone and found they act synergistically to increase SLPI transcript levels. Our findings suggest that corticosteroids may exert their antiinflammatory effects in part by increasing airway epithelial cell SLPI production.