Inhibition of phosphodiesterase activity, airway inflammation and hyperresponsiveness by PDE4 inhibitor and glucocorticoid in a murine model of allergic asthma

Phosphodiesterase 4 (PDE4) isozyme plays important roles in inflammatory and immunomodulatory cells. In this study, piclamilast, a selective PDE4 inhibitor, was used to investigate the role of PDE4 in respiratory function and inflammation in a murine asthma model. Sensitized mice were challenged with aerosolized ovalbumin for 7 days, piclamilast (1, 3 and 10 mg/kg) and dexamethasone (2 mg/kg) were orally administered once daily during the period of challenge. Twenty-four hours after the last challenge, airway hyperresponsiveness to methacholine was determined by whole-body plethysmography, airway inflammation and mucus secretion by histomorphometry, pulmonary cAMP-PDE activity by HPLC, cytokine levels in bronchoalveolar lavage fluid and their mRNA expression in lung by ELISA and RT-PCR, respectively. In control mice, significant induction of cAMP-PDE activity was parallel to the increases of hyperresponsiveness, inflammatory cells, cytokine levels, mRNA expression as well as goblet cell hyperplasia. However, piclamilast dose-dependently and significantly improved airway resistance and dynamic compliance, and the maximal effect was similar to that of dexamethasone. Piclamilast treatment dose-dependently and significantly prevented the increase in inflammatory cell number and goblet cell hyperplasia, as well as production of cytokines, including eotaxin, TNFalpha and IL-4. Piclamilast exerted a weaker inhibitory effect than dexamethasone on eosinophils and neutrophils, had no effect on lymphocyte accumulation. Moreover, piclamilast inhibited up-regulation of cAMP-PDE activity and cytokine mRNA expression; the maximal inhibition of cAMP-PDE was greater than that exerted by dexamethasone, and was similar to dexamethasone on cytokine mRNA expression. This study suggests that inhibition of PDE4 by piclamilast robustly improves the pulmonary function, airway inflammation and goblet cell hyperplasia in murine allergenic asthma.     

Secretory leukocyte protease inhibitor plays an important role in the regulation of allergic asthma in mice

Secretory leukocyte protease inhibitor (SLPI) is an anti-inflammatory protein that is observed at high levels in asthma patients. Resiquimod, a TLR7/8 ligand, is protective against acute and chronic asthma, and it increases SLPI expression of macrophages in vitro. However, the protective role played by SLPI and the interactions between the SLPI and resiquimod pathways in the immune response occurring in allergic asthma have not been fully elucidated. To evaluate the role of SLPI in the development of asthma phenotypes and the effect of resiquimod treatment on SLPI, we assessed airway resistance and inflammatory parameters in the lungs of OVA-induced asthmatic SLPI transgenic and knockout mice and in mice treated with resiquimod. Compared with wild-type mice, allergic SLPI transgenic mice showed a decrease in lung resistance (p < 0.001), airway eosinophilia (p < 0.001), goblet cell hyperplasia (p < 0.001), and plasma IgE levels (p < 0.001). Allergic SLPI knockout mice displayed phenotype changes significantly more severe compared with wild-type mice. These phenotypes included lung resistance (p < 0.001), airway eosinophilia (p < 0.001), goblet cell hyperplasia (p < 0.001), cytokine levels in the lungs (p < 0.05), and plasma IgE levels (p < 0.001). Treatment of asthmatic transgenic mice with resiquimod increased the expression of SLPI and decreased inflammation in the lungs; resiquimod treatment was still effective in asthmatic SLPI knockout mice. Taken together, our study showed that the expression of SLPI protects against allergic asthma phenotypes, and treatment by resiquimod is independent of SLPI expression, displayed through the use of transgenic and knockout SLPI mice.     

DA-9601, Artemisia asiatica herbal extract, ameliorates airway inflammation of allergic asthma in mice

We previously reported that DA-9601, ethanol herbal extract of Artemisia asiatica, inhibited histamine and leukotriene releases in guinea pig lung mast cells activated with specific antigen/antibody reaction. This study aimed to evaluate the inhibitory effect of DA-9601 on the OVA-induced airway inflammation in allergic asthma mouse model. BALB/c mice were sensitized and challenged with OVA. DA-9601 was administered orally 1 h before every local OVA-challenge. OVA-specific serum IgE was measured by ELISA, recruitment of inflammatory cells in BAL fluids and lung tissues by Diff-Quik and H&E staining, respectively, the expressions of CD40, CD40L and VCAM-1 by immunohistochemistry, goblet cell hyperplasia by PAS staining, activities of MMPs by gelatin zymography, expressions of mRNA and proteins of cytokines by RT-PCR and ELISA, activities of MAP kinases by western blot, and activity of NF-KappaB by EMSA. DA-9601 reduced IgE level, recruitment of inflammatory cells into the BAL fluid and lung tissues, expressions of CD40, CD40L and VCAM-1 molecules, goblet cell hyperplasia, MMPs activity, expressions of mRNA and productions of various cytokines, activities of MAP kinases and NK-KappaB increased from OVA-challenged mice. These data suggest that DA-9601 may be developed as a clinical therapeutic agent in allergic diseases due to suppressing the airway allergic inflammation via regulation of various cellular molecules expressed by MAP kinases/NF-KappaB pathway.     

Effect of phoshpodiesterase 4 (PDE4) inhibibtors on eotaxin expression in humen bronchial epithelial cells

The increasing number of eosinophils into bronchoaelvolar space is observed during noninfectious inflammatory lung diseases. Eotaxins (eotaxin-1/CCL11, eotaxin-2/CCL24, eotaxin-3/CCL26) are the strongest chemotactic agents for eosinophils. Inhibitors of phosphodiesterase 4 (PDE4), the enzyme decomposing cAMP, are anti-inflammatory agents which act through cAMP elevation and inhibit numerous steps of allergic inflammation. The effect of PDE4 inhibitors on eotaxin expression is not known in details. The aim of our study was to evaluate the influence of PDE4 inhibitors: rolipram and RO-20-1724 on expression of eotaxins in bronchial epithelial cell line BEAS-2B. Cells were preincubated with PDE4 inhibitors or dexamethasone for 1 hour and then stimulated with IL-4 or IL-13 alone or in combination with TNF-α. After 48 hours eotaxin protein level was measured by ELISA and mRNA level by real time PCR. Results: PDE4 inhibitors decreased CCL11 and CCL26 expression only in cultures co-stimulated with TNF-α. In cultures stimulated with IL-4 and TNF-α rolipram and RO-20-1724 diminished CCL11 mRNA expression by 34 and 37%, respectively, and CCL26 by 43 and 47%. In cultures stimulated with IL-13 and TNF-α rolipram and RO-20-1724 decreased expression of both eotaxins by about 50%. These results were confirmed at the protein level. The effect of PDE4 inhibitors on eotaxin expression in BEAS-2B cells, in our experimental conditions, depends on TNF-α contribution.     

Antibody to mCLCA3 Suppresses Symptoms in a Mouse Model of Asthma

Background

Asthma is a complex and heterogeneous chronic inflammatory disorder that is associated with mucous cell metaplasia and mucus hypersecretion. Functional genomic analysis indicates that mucous cell metaplasia and mucus hypersecretion depend on members of the calcium-activated chloride channel (CLCA) gene family. It has been reported that the inhibition of CLCAs could relieve the symptoms of asthma. Thus, the mCLCA3 antibody may be a promising strategy to treat allergic diseases such as asthma.

Methods

We constructed asthmatic mouse models of OVA-induced chronic airway inflammatory disorder to study the function of the mCLCA3 antibody. Airway inflammation was measured by HE staining; goblet cell hyperplasia and mucus hypersecretion were detected by PAS staining; muc5ac, IL-13, IFN-γ levels in bronchoalveolar lavage fluid (BALF) were examined by ELISA; Goblet cell apoptosis was measured by TUNEL assay and alcian blue staining; mCLCA3, Bcl-2 and Bax expression were detected by RT-PCR, Western blotting and immunohistochemical analysis.

Results

In our study, mice treated with mCLCA3 antibody developed fewer pathological changes compared with control mice and asthmatic mice, including a remarkable reduction in airway inflammation, the number of goblet cells and mCLCA3 expression in lung tissue. The levels of muc5ac and IL-13 were significantly reduced in BALF. We also found that the rate of goblet cell apoptosis was increased after treatment with mCLCA3 antibody, which was accompanied by an increase in Bax levels and a decrease in Bcl-2 expression in goblet cells.

Conclusions

Taken together, our results indicate that mCLCA3 antibody may have the potential as an effective pharmacotherapy for asthma.     

NCS 613, a potent and specific PDE4 inhibitor, displays anti-inflammatory effects on human lung tissues

Chronic inflammation is a hallmark of pulmonary diseases, which leads to lung parenchyma destruction (emphysema) and obstructive bronchiolitis occurring in both chronic obstructive pulmonary disease and asthma. Inflammation is strongly correlated with low intracellular cAMP levels and increase in specific cAMP hydrolyzing activity. The aim of the present study was to investigate the role of the cyclic phosphodiesterase type 4 (PDE4) in human lung and to determine the effects of NCS 613, a new PDE4 inhibitor, on lung inflammation and bronchial hyperresponsiveness. High cAMP-PDE activities were found in the cytosoluble fractions from human lung parenchyma and distal bronchi. PDE4 (rolipram sensitive) represented 40% and 56% of total cAMP-PDE activities in the above-corresponding tissues. Moreover, PDE4A, PDE4B, PDE4C, and PDE4D isoforms were detected in all three subcellular fractions (cytosolic, microsomal, and nuclear) with differential distributions according to specific variants. Pharmacological treatments with NCS 613 significantly decreased PDE4 activity and reduced IκBα degradation in cultured parenchyma, both of which are usually correlated with a lower inflammation status. Moreover, NCS 613 pretreatment potentiated isoproterenol-induced relaxations in human distal bronchi, while reducing TNF-α-induced hyperresponsiveness in cultured bronchi, as assessed in the presence of methacholine, U-46619, or histamine. This reducing effect of NCS 613 on human bronchi hyperresponsiveness triggered by TNF-α was related to a lower expression level of PDE4B and PDE4C, as well as a downregulation of the phosphorylated forms of p38-MAPK, CPI-17, and MYPT-1, which are known to control tone. In conclusion, specific PDE4 inhibitors, such as NCS 613, may represent an alternative and isoform-specific approach toward reducing human lung inflammation and airway overreactivity.     

Neutrophil elastase induces mucus cell metaplasia in mouse lung

Goblet cell hyperplasia in the superficial airway epithelia is a signature pathological feature of chronic bronchitis and cystic fibrosis. In these chronic inflammatory airway diseases, neutrophil elastase (NE) is found in high concentrations in the epithelial lining fluid. NE has been reported to trigger mucin secretion and increase mucin gene expression in vitro. We hypothesized that chronic NE exposure to murine airways in vivo would induce goblet cell metaplasia. Human NE (50 microg) or PBS saline was aspirated intratracheally by male Balb/c (6 wk of age) mice on days 1, 4, and 7. On days 8, 11, and 14, lung tissues for histology and bronchoalveolar lavage (BAL) samples for cell counts and cytokine levels were obtained. NE induced Muc5ac mRNA and protein expression and goblet cell metaplasia on days 8, 11, and 14. These cellular changes were the result of proteolytic activity, since the addition of an elastase inhibitor, methoxysuccinyl Ala-Ala-Pro-Val chloromethylketone (AAPV-CMK), blocked NE-induced Muc5ac expression and goblet cell metaplasia. NE significantly increased keratinocyte-derived chemokine and IL-5 in BAL and increased lung tissue inflammation and BAL leukocyte counts. The addition of AAPV-CMK reduced these measures of inflammation to control levels. These experiments suggest that NE proteolytic activity initiates an inflammatory process leading to goblet cell metaplasia.     

Resistin-like molecule-beta is an allergen-induced cytokine with inflammatory and remodeling activity in the murine lung

Resistin-like molecule (RELM)-beta is a cysteine-rich cytokine implicated in insulin resistance and asthmatic responses, but its function remains an enigma. We now report that RELM-beta has a role in promoting airway inflammation and lung remodeling in the mouse lung. RELM-beta is strongly induced by diverse allergens and T helper type 2 (Th2) cytokines by an IL-13- and STAT6-dependent mechanism. To understand the in vivo role of RELM-beta, we delivered recombinant murine RELM-beta intratracheally to na?ve mice. RELM-beta induced dose-dependent leukocyte accumulation (most prominently involving macrophages) and goblet cell hyperplasia. The most prominent effect induced by RELM-beta was increased perivascular and peribronchial collagen deposition. Mice genetically deficient in RELM-beta had reduced accumulation of collagen and goblet cell hyperplasia in an experimental model of allergic airway inflammation. In vitro experiments demonstrated that RELM-beta had fibroblast motogenic activity. These results identify RELM-beta as a Th2-associated cytokine with potent inflammatory and remodeling activity.     

Altered allergen-induced eosinophil trafficking and physiological dysfunction in airways with preexisting virus-induced injury

Although both asthmatics and allergic rhinitics develop an acute inflammatory response to lower airway allergen challenge, only asthmatics experience airway obstruction resulting from chronic environmental allergen exposure. Hypothesizing that asthmatic airways have an altered response to chronic allergic inflammation, we compared the effects of repeated low-level exposures to inhaled Alternaria extract in sensitized rats with preexisting chronic postbronchiolitis airway dysfunction versus sensitized controls with normal airways. Measurements of air space (bronchoalveolar lavage) inflammatory cells, airway goblet cells, airway wall collagen, airway wall eosinophils, airway alveolar attachments, and pulmonary physiology were conducted after six weekly exposures to aerosolized saline or Alternaria extract. Postbronchiolitis rats, but not those starting with normal airways, had persistent increases in airway wall eosinophils, goblet cell hyperplasia in small airways, and loss of lung elastic recoil after repeated exposure to aerosolized Alternaria extract. Despite having elevated airway wall eosinophils, the postbronchiolitis rats had no eosinophils in bronchoalveolar lavage at 5 days after the last allergen exposure, suggesting altered egression of tissue eosinophils into the air space. In conclusion, rats with preexisting airway pathology had altered eosinophil trafficking and allergen-induced changes in airway epithelium and lung mechanics that were absent in sensitized control rats that had normal airways before the allergen exposures.     

Protective Effects of the Polyphenol Sesamin on Allergen-Induced TH2 Responses and Airway Inflammation in Mice

Allergic asthma is a lifelong airway condition that affects people of all ages. In recent decades, asthma prevalence continues to increase globally, with an estimated number of 250,000 annual deaths attributed to the disease. Although inhaled corticosteroids and β-adrenergic receptor agonists are the primary therapeutic avenues that effectively reduce asthma symptoms, profound side effects may occur in patients with long-term treatments. Therefore, development of new therapeutic strategies is needed as alternative or supplement to current asthma treatments. Sesamin is a natural polyphenolic compound with strong anti-oxidative effects. Several studies have reported that sesamin is effective in preventing hypertension, thrombotic tendency, and neuroinflammation. However, it is still unknown whether sesamin can reduce asthma-induced allergic inflammation and airway hyperresponsiveness (AHR). Our study has revealed that sesamin exhibited significant anti-inflammatory effects in ovalbumin (OVA)-induced murine asthma model. We found that treatments with sesamin after OVA sensitization and challenge significantly decreased expression levels of interleukin-4 (IL-4), IL-5, IL-13, and serum IgE. The numbers of total inflammatory cells and eosinophils in BALF were also reduced in the sesamin-treated animals. Histological results demonstrated that sesamin attenuated OVA-induced eosinophil infiltration, airway goblet cell hyperplasia, mucus occlusion, and MUC5AC expression in the lung tissue. Mice administered with sesamin showed limited increases in AHR compared with mice receiving vehicle after OVA challenge. OVA increased phosphorylation levels of IκB-α and nuclear expression levels of NF-κB, both of which were reversed by sesamin treatments. These data indicate that sesamin is effective in treating allergic asthma responses induced by OVA in mice.     

Anti-inflammatory effects of mitogen-activated protein kinase kinase inhibitor U0126 in an asthma mouse model

Mitogen-activated protein kinase (MAPK) signaling cascade plays a pivotal role in the activation of inflammatory cells. Recent findings revealed that the activity of p42/44 MAPK (also known as extracellular signal-regulated kinase (ERK)) in the lungs was significantly higher in asthmatic mice than in normal controls. We hypothesized that inhibition of ERK activity may have anti-inflammatory effects in allergic asthma. BALB/c mice were sensitized with OVA and, upon OVA aerosol challenge, developed airway eosinophilia, mucus hypersecretion, elevation in cytokine and chemokine levels, up-regulation of VCAM-1 expression, and airway hyperresponsiveness. Intraperitoneal administration of U0126, a specific MAPK/ERK kinase inhibitor, significantly (p < 0.05) inhibited OVA-induced increases in total cell counts, eosinophil counts, and IL-4, IL-5, IL-13, and eotaxin levels recovered in bronchoalveolar lavage fluid in a dose-dependent manner. U0126 also substantially (p < 0.05) reduced the serum levels of total IgE and OVA-specific IgE and IgG1. Histological studies show that U0126 dramatically inhibited OVA-induced lung tissue eosinophilia, airway mucus production, and expression of VCAM-1 in lung tissues. In addition, U0126 significantly (p < 0.05) suppressed OVA-induced airway hyperresponsiveness to inhaled methacholine in a dose-dependent manner. Western blot analysis of whole lung lysates shows that U0126 markedly attenuated OVA-induced tyrosine phosphorylation of ERK1/2. Taken together, our findings implicate that inhibition of ERK signaling pathway may have therapeutic potential for the treatment of allergic airway inflammation.     

Inhibition of allergic inflammation in the airways using aerosolized antisense to Syk kinase

Activation of the protein tyrosine kinase Syk is an early event that follows cross-linking of Fc gamma R and Fc epsilon R, leading to the release of biologically active molecules in inflammation. We reported previously that aerosolized Syk antisense oligodeoxynucleotides (ASO) depresses Syk expression in inflammatory cells, the release of mediators from alveolar macrophages, and pulmonary inflammation. To study the effect of Syk ASO in allergic inflammation and airway hyperresponsiveness, we used the Brown Norway rat model of OVA-induced allergic asthma. Syk ASO, delivered in a liposome, carrier/lipid complex by aerosol to rats, significantly inhibited the Ag-induced inflammatory cell infiltrate in the bronchoalveolar space, decreasing both neutrophilia and eosinophilia. The number of eosinophils in the lung parenchyma was also diminished. Syk ASO also depressed up-regulation of the expression of beta(2) integrins, alpha(4) integrin, and ICAM-1 in bronchoalveolar lavage leukocytes and reversed the Ag-induced decrease in CD62L expression on neutrophils. Furthermore, the increase in TNF levels in bronchoalveolar lavage following Ag challenge was significantly inhibited. Syk ASO also suppressed Ag-mediated contraction of the trachea in a complementary model. Thus, aerosolized Syk ASO suppresses many of the central components of allergic asthma and inflammation and may provide a new therapeutic approach.     

Clonorchis sinensis-derived total protein attenuates airway inflammation in murine asthma model by inducing regulatory T cells and modulating dendritic cell functions

Asthma is characterized by Th2-mediated inflammation, resulting in airway hyperresponsiveness (AHR) through airway remodeling. Recent epidemiological and experimental reports have suggested an inverse relationship between the development of allergy and helminth infections. Infection by Clonorchis sinensis, a liver fluke that resides in the bile duct of humans, is endemic predominantly in Asia including Korea and China. Using a murine model for asthma, we investigated the effects of C. sinensis-derived total protein (Cs-TP) on allergen-induced airway inflammation and the mechanism underlying the protective effects of Cs-TP administration on asthma. Treatment with Cs-TP attenuated OVA-induced airway inflammation and methacholine-induced AHR, as well as eosinophilia development, lymphocyte infiltration into the lung, and goblet cell metaplasia. This protective effect of Cs-TP is associated with markedly reduced OVA-specific IgE and Th1/Th2 cytokine production. Moreover, Cs-TP increased the number of CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells as well as their suppressive activity. In fact, proliferation of OVA-restimulated splenocytes was suppressed significantly. Cs-TP also inhibited the expression of such co-stimulatory molecules as CD80, CD86, and CD40 in LPS- or OVA-stimulated dendritic cells (DCs), suggesting that Cs-TP could interfere with the capacity of airway DCs to prime naïve T cells. These data demonstrate the capacity of C. sinensis to ameliorate allergic asthma and broaden our understanding of the paradoxical relationship between the allergic immune response and helminth infection.     

Altered activities of cyclic nucleotide phosphodiesterases and soluble guanylyl cyclase in cultured RFL-6 cells

We utilized rat fetal lung fibroblasts (RFL-6) to evaluate our PDE5 inhibitors at cellular level and observed a decrease in cGMP accumulation induced by sodium nitroprusside (SNP) and PDE5 inhibitors with passage. To further investigate this observation, we examined cGMP synthesis via soluble guanylyl cyclase (sGC) and degradation via phosphodiesterases (PDEs) at different passages. At passage (p)4, p9, p14, major cGMP and cAMP degradation activities were contributed by PDE5 and PDE4, respectively. The PDE5 activity decreased 50% from p4 to p14, while PDE4 activity doubled. The cGMP accumulation was evaluated in the presence of sodium nitroprusside (SNP) and/or PDE inhibitors in p4 and p14 cells. SNP together with sildenafil, a PDE5 inhibitor, induced dose-dependent increase in cGMP levels in cells at p4, but showed little effect on cells at p14. The possible down regulation of sGC at mRNA level was explored using real-time RT-PCR. The result showed the mRNA level of the alpha1 subunit of sGC decreased about 98% by p9, while the change on beta1 mRNA was minimal. Consistently, sGC activities in cell lysate decreased by 94% at p9. Forskolin stimulated a dramatic increase in cAMP levels in cells at all passages examined. Our results show that sGC activity decreased significantly and rapidly with passage due to a down regulation of the alpha1 subunit mRNA, yet the adenylyl cyclase activity was not compromised. This study further emphasized the importance of considering passage number when using cell culture as a model system to study NO/cGMP pathway.     

Osthol attenuates neutrophilic oxidative stress and hemorrhagic shock-induced lung injury via inhibition of phosphodiesterase 4

Oxidative stress caused by neutrophils is an important pathogenic factor in trauma/hemorrhagic (T/H)-induced acute lung injury (ALI). Osthol, a natural coumarin found in traditional medicinal plants, has therapeutic potential in various diseases. However, the pharmacological effects of osthol in human neutrophils and its molecular mechanism of action remain elusive. In this study, our data showed that osthol potently inhibited the production of superoxide anion (O₂^•−) and reactive oxidants derived therefrom as well as expression of CD11b in N-formylmethionylleucylphenylalanine (FMLP)-activated human neutrophils. However, osthol inhibited neutrophil degranulation only slightly and it failed to inhibit the activity of subcellular NADPH oxidase. FMLP-induced phosphorylation of extracellular signal-regulated kinase (ERK) and protein kinase B (Akt) was inhibited by osthol. Notably, osthol increased the cAMP concentration and protein kinase A (PKA) activity in activated neutrophils. PKA inhibitors reversed the inhibitory effects of osthol, suggesting that these are mediated through cAMP/PKA-dependent inhibition of ERK and Akt activation. Furthermore, the activity of cAMP-specific phosphodiesterase (PDE) 4, but not PDE3 or PDE7, was significantly reduced by osthol. In addition, osthol reduced myeloperoxidase activity and pulmonary edema in rats subjected to T/H shock. In conclusion, our data suggest that osthol has effective anti-inflammatory activity in human neutrophils through the suppression of PDE4 and protects significantly against T/H shock-induced ALI in rats. Osthol may have potential for future clinical application as a novel adjunct therapy to treat lung inflammation caused by adverse circulatory conditions.     

Nicotine primarily suppresses lung Th2 but not goblet cell and muscle cell responses to allergens

Allergic asthma, an inflammatory disease characterized by the infiltration and activation of various leukocytes, the production of Th2 cytokines and leukotrienes, and atopy, also affects the function of other cell types, causing goblet cell hyperplasia/hypertrophy, increased mucus production/secretion, and airway hyperreactivity. Eosinophilic inflammation is a characteristic feature of human asthma, and recent evidence suggests that eosinophils also play a critical role in T cell trafficking in animal models of asthma. Nicotine is an anti-inflammatory, but the association between smoking and asthma is highly contentious and some report that smoking cessation increases the risk of asthma in ex-smokers. To ascertain the effects of nicotine on allergy/asthma, Brown Norway rats were treated with nicotine and sensitized and challenged with allergens. The results unequivocally show that, even after multiple allergen sensitizations, nicotine dramatically suppresses inflammatory/allergic parameters in the lung including the following: eosinophilic/lymphocytic emigration; mRNA and/or protein expression of the Th2 cytokines/chemokines IL-4, IL-5, IL-13, IL-25, and eotaxin; leukotriene C(4); and total as well as allergen-specific IgE. Although nicotine did not significantly affect hexosaminidase release, IgG, or methacholine-induced airway resistance, it significantly decreased mucus content in bronchoalveolar lavage; interestingly, however, despite the strong suppression of IL-4/IL-13, nicotine significantly increased the intraepithelial-stored mucosubstances and Muc5ac mRNA expression. These results suggest that nicotine modulates allergy/asthma primarily by suppressing eosinophil trafficking and suppressing Th2 cytokine/chemokine responses without reducing goblet cell metaplasia or mucous production and may explain the lower risk of allergic diseases in smokers. To our knowledge this is the first direct evidence that nicotine modulates allergic responses.     

Lixazinone stimulates mitogenesis of Madin-Darby canine kidney cells

Polycystic kidney diseases (PKD) are characterized by excessive proliferation of renal tubular epithelial cells, development of fluid-filled cysts, and progressive renal insufficiency. cAMP inhibits proliferation of normal renal tubular epithelial cells but stimulates proliferation of renal tubular epithelial cells derived from patients with PKD. Madin-Darby canine kidney (MDCK) epithelial cells, which are widely used as an in vitro model of cystogenesis, also proliferate in response to cAMP. Intracellular cAMP levels are tightly regulated by phosphodiesterases (PDE). Isoform-specific PDE inhibitors have been developed as therapeutic agents to regulate signaling pathways directed by cAMP. In other renal cell types, we have previously demonstrated that cAMP is hydrolyzed by PDE3 and PDE4, but only PDE3 inhibitors suppress proliferation by inhibiting Raf-1 activity (Cheng J, Thompson MA, Walker HJ, Gray CE, Diaz Encarnacion MM, Warner GM, Grande JP. Am J Physiol Renal Physiol 287:F940-F953, 2004.) A potential role for PDE isoform(s) in cAMP-mediated proliferation of MDCK cells has not previously been established. Similar to what we have previously found in several other renal cell types, cAMP hydrolysis in MDCK cells is directed primarily by PDE4 (85% of total activity) and PDE3 (15% of total activity). PDE4 inhibitors are more effective than PDE3 inhibitors in increasing intracellular cAMP levels in MDCK cells. However, only PDE3 inhibitors, and not PDE4 inhibitors, stimulate mitogenesis of MDCK cells. PDE3 but not PDE4 inhibitors activate B-Raf but not Raf-1, as assessed by an in vitro kinase assay. PDE3 but not PDE4 inhibitors activate the ERK pathway and activate cyclins D and E, as assessed by histone H1 kinase assay. We conclude that mitogenesis of MDCK cells is regulated by a functionally compartmentalized intracellular cAMP pool directed by PDE3. Pharmacologic agents that stimulate PDE3 activity may provide the basis for new therapies directed toward reducing cystogenesis in patients with PKD.     

Inhibition of phosphodiesterase 4 amplifies cytokine-dependent induction of arginase in macrophages

Arginase is greatly elevated in asthma and is thought to play a role in the pathophysiology of this disease. As inhibitors of phosphodiesterase 4 (PDE4), the predominant PDE in macrophages, elevate cAMP levels and reduce inflammation, they have been proposed for use in treatment of asthma and chronic obstructive pulmonary disease. As cAMP is an inducer of arginase, we tested the hypothesis that a PDE4 inhibitor would enhance macrophage arginase induction by key cytokines implicated in asthma and other pulmonary diseases. RAW 264.7 cells were stimulated with IL-4 or transforming growth factor (TGF)-beta, with and without the PDE4 inhibitor rolipram. IL-4 and TGF-beta increased arginase activity 16- and 5-fold, respectively. Rolipram alone had no effect but when combined with IL-4 and TGF-beta synergistically enhanced arginase activity by an additional 15- and 5-fold, respectively. The increases in arginase I protein and mRNA levels mirrored increases in arginase activity. Induction of arginase II mRNA was also enhanced by rolipram but to a much lesser extent than arginase I. Unlike its effect in RAW 264.7 cells, IL-4 alone did not increase arginase activity in human alveolar macrophages (AM) from healthy volunteers. However, combining IL-4 with agents to induce cAMP levels induced arginase activity in human AM significantly above the level obtained with cAMP-inducing agents alone. In conclusion, agents that elevate cAMP significantly enhance induction of arginase by cytokines. Therefore, consequences of increased arginase expression should be evaluated whenever PDE inhibitors are proposed for treatment of inflammatory disorders in which IL-4 and/or TGF-beta predominate.     

Complex role of the IL-4 receptor alpha in a murine model of airway inflammation: expression of the IL-4 receptor alpha on nonlymphoid cells of bone marrow origin contributes to severity of inflammation

Recent studies have suggested the IL-4Ralpha expressed on lung epithelium is necessary for TH2-mediated goblet cell differentiation and mucus hypersecretion in a murine model of allergic lung disease. However, the IL-4Ralpha is expressed on numerous cell types that could contribute to the overall pathology and severity of asthma. The relative role of the receptor on these cells has not yet been conclusively delineated. To dissect the contribution of IL-4Ralpha in the development of pulmonary allergic responses, we generated murine radiation bone marrow (BM) chimeras. BM from IL-4Ralpha(+) or IL-4Ralpha(-) mice was transferred into recipient mice that expressed or lacked IL-4Ralpha. In the absence of IL-4Ralpha in recipient mice, there was no goblet cell metaplasia or mucus hypersecretion in response to OVA, even in the presence of TH2 cells and substantial eosinophilic infiltration. More importantly, we found that expression of the IL-4Ralpha on a nonlymphoid, MHC class II(+), BM-derived cell type contributes to the severity of inflammation and mucus production. These results suggest that IL-4 and IL-13 contribute to the development of allergic inflammation by stimulating a complex interaction between IL-4Ralpha(+) cell types of both bone marrow and non-bone marrow origin.