articleEffects of phosphodiesterase 4 inhibition on bleomycin-induced pulmonary fibrosis in mice

Background: Pulmonary fibrosis (PF) is a group of devastating and largely irreversible diseases. Phosphodiesterase (PDE) 4 is involved in the processes of remodeling and inflammation, which play key role in tissue fibrosis. The aim of the study was, therefore, to investigate the effect of PDE4 inhibition in experimental model of PF. Methods: PF was induced in C57BL/6N mice by instillation of bleomycin. Pharmacological inhibition of PDE4 was achieved by using cilomilast, a selective PDE4 inhibitor. Changes in either lung inflammation or remodeling were evaluated at different stages of experimental PF. Lung inflammation was assessed by bronchoalveolar lavage fluid (BALF) differential cell count and reverse transcription quantitative polymerase chain reaction (RT-qPCR) for inflammatory cytokines. Changes in tissue remodeling were evaluated by pulmonary compliance measurement, quantified pathological examination, measurement of collagen deposition and RT-qPCR for late remodeling markers. Survival in all groups was analyzed as well. Results: PDE4 inhibition significantly reduced the total number of alveolar inflammatory cells in BALF of mice with bleomycin-induced PF at early fibrosis stage (days 4 and 7). Number of macrophages and lymphocytes, but not neutrophils, was significantly reduced as well. Treatment decreased lung tumor necrosis factor (TNF)-α mRNA level and increased mRNA level of interleukin (IL)-6 but did not influence IL-1β. At later stage (days 14 and 24) cilomilast improved lung function, which was shown by increase in lung compliance. It also lowered fibrosis degree, as was shown by quantified pathological examination of Hematoxilin-Eosin stained lung sections. Cilomilast had no significant effect on the expression of late remodeling markers such as transforming growth factor (TGF)-β1 and collagen type Ia1 (COL(I)α1). However, it tended to restore the level of lung collagen, assessed by SIRCOL assay and Masson's trichrome staining, and to improve the overall survival. Conclusions: Selective PDE4 inhibition suppresses early inflammatory stage and attenuates the late stage of experimental pulmonary fibrosis. Background Pulmonary fibrosis represents a group of devastating and largely irreversible human interstitial lung diseases having only limited treatment options. The disease is characterized by chronic interstitial inflammation, abnormal function of interstitial fibroblasts and deposition of excessive amounts of collagen, altogether leading to severe tissue remodeling [1]. Pathological changes are accompanied by elevated expression of cytokines TNF-α, IL-1β, IL-6, growth factors and matrix metalloproteases (MMPs) [2-4]. The most common experimental model of human PF is bleomycin-induced PF in mice. It is characterized by inflammatory and remodeling stages, which allows studying different aspects of the disease [5]. Phosphodiesterases are a superfamily of enzymes that hydrolyze cAMP and/or cGMP and thereby regulate the intracellular levels of second messengers [6]. Members of PDE4 family (E.C. 3.1.4.17) are cAMP-specific PDEs composed of number of isoforms and are highly represented in the lung [7-10]. As a component of cAMP/protein kinase A (PKA) pathway PDE4 plays direct role in * Correspondence: ralph.schermuly@mpi-bn.mpg.de 1 University of Giessen, Department of Internal Medicine, Giessen, Germany Full list of author information is available at the end of the article BioMed Central © 2010 Udalov et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Udalov et al. BMC Pulmonary Medicine 2010, 10:26 http://www.biomedcentral.com/1471-2466/10/26 Page 2 of 9 cell proliferation, differentiation and migration through regulation of the cAMP level [11-13]. Finally, PDE4 represents the major cAMP-hydrolyzing enzyme in monocytes, lymphocytes and neutrophils and its activation is required for inflammatory response [8,14,15]. For these reasons, PDE4 inhibitors were suggested for treatment of several lung diseases as new anti-inflammatory and anti-remodeling agents [16]. Our group has previously demonstrated that PDE3/4 inhibitor attenuates migration of pulmonary artery smooth muscle cells (PASMCs) in vitro and reverses pulmonary vascular remodeling in vivo [17]. PDE4 inhibitor cilomilast was also shown to suppress the release and activation of MMP-1 and MMP-9 from lung fibroblasts, which are known to be involved in PF progression [18]. Furthermore, cilomilast and other PDE4 inhibitors were demonstrated to inhibit lung TNF-α and TGF-β release, as well as neutrophil influx in vivo [19-21]. Finally, treatment of experimental chronic colitis with PDE4 inhibitor rolipram resulted in decreased collagen deposition as well as TNF-α and TGF-β content in the tissue [22]. In the present study we hypothesized that PDE4 inhibitors are able to modulate both inflammatory response and tissue remodeling. The aim of the study was, therefore, to investigate the effects of selective PDE4 inhibitor on different aspects of experimental PF in vivo. Methods Animals Adult male 5-6 weeks-old C57BL/6N mice weighting 1921 g were obtained from Charles River Laboratories (Germany). Animals were housed under room temperature and 12/12-hour light/dark cycle with free access to food and water. All experiments were performed in accordance with the National Institutes of Health Guidelines on the Use of Laboratory Animals. Both the University Animal Care Committee and the Federal Authorities for Animal Research of the Regierungspraesidium Giessen (Hessen, Germany) approved the study protocol. Bleomycin administration and treatment groups At day 0 mice were given anesthesia with isofluran (Baxter, Germany) followed by random orotracheal instillation of bleomycin (Sigma, Germany) or sterile saline (0.9% NaCl) with the mouse nose kept pinched. Bleomycin dissolved in sterile saline was given at the dose of 2.8 units/kg. Animals were assigned to the following groups 1) "saline", 2) "bleo+ctrl" and 3) "bleo+cilo". "Saline" group received instillation of sterile saline at day 0 and was given vehicle alone (2% aqueous methylcellulose solution). Mice in "bleo+ctrl" group received instillation of bleomycin at day 0 and were given vehicle alone. Mice in "bleo+cilo" group received instillation of bleomycin at day 0 and were treated once a day with 50 mg/kg cilomilast (SB207499 or Ariflo®, [c-4-cyano-4-(3-cyclopentyloxy-4methoxyphenyl)-r-l-cyclohexane carboxylic acid]) (Nycomed, Germany), suspended in vehicle. Solutions were administered per os via feeding needle, all in the same manner. Treatment in all groups started at day 0 and lasted till the end of experiment, i.e. for 4, 7, 14 or 24 days. Bronchoalveolar lavage fluid cell count At days 4 and 7 after bleomycin instillation mice were euthanized by injecting a lethal dose of pentobarbital. Lungs were flushed three times with 0.5 ml ice cold PBSEDTA, recovered fluid was centrifuged and cell pellet was re-suspended in 1 ml of ice-cold saline. Total cell count was performed using Neubauer counting chamber (depth 0.1 mm, 0.0025 mm2; Optik Labor, Germany). For differential cell count cells in constant volume of 0.2 ml of PBS were transferred to a glass slide with Cytospin-3 centrifuge (Shandon Scientific Ltd, UK) and stained with May Gruenwald/Giemsa. Numbers of macrophages, neutrophils and lymphocytes were determined by counting on light microscope (Q550IW; Leica, Germany) among 100 of total cells. These data were then extrapolated to number of cells per milliliter. Lung compliance and histological examination At days 14 and 24 after bleomycin instillation mice were subjected to lung compliance measurement as described previously [23]. Briefly, animals were anesthetized with i.p. injection of ketamin/xylacinehydrochloride (Bayer, Germany). Trachea was canulated, mice were placed in the chamber and connected to the instrument. Volume of 0.3 ml and pressure of 3 kPa were set for calculating compliance as a ratio of volume to pressure (ml/kPa). Lungs of mice were isolated at the same time points. Four right lobes were inflated with 4.5% formaldehyde solution at constant pressure and fixated as described elsewhere. After dehydration in tissue processor lung lobes were individually embedded in paraffin, sectioned at 3 μm on microtome (all instruments from Leica, Germany), mounted on glass slides and stained either with Hematoxilin-Eosin or Masson's trichrome according to standard protocols. For histological assessment, HematoxilinEosin-stained slides were scanned with light microscope (Leica, Germany) at 100× magnification yielding 50-100 images for each lobe (up to 300 per animal). Each of the images was reviewed and degree of fibrosis was assigned according to Ashcroft's fibrosis score system [24] with slight modifications: normal lung was referred to as score "0" while score "6" represented maximal degree of pathological changes. Collagen assay Level of collagen in lung tissues was determined by SIRCOL collagen assay (Biocolor Ltd., UK) according to manufacturer's instructions. Briefly, left lung lobes were Udalov et al. BMC Pulmonary Medicine 2010, 10:26 http://www.biomedcentral.com/1471-2466/10/26 Page 3 of 9 homogenized and collagen was solubilized in 0.5 M acetic acid. Extracts were incubated with Sirius red dye and absorbance was determined at 540 nm with spectrophotometer Infinite M200 (Tecan, Austria). Amount of collagen was expressed in μg/g of wet tissue. Survival analysis Survival of mice for each treatment group was expressed in percent of animals left of original number at the specific time points of the experiment. Mice were followed up for 24 days. RNA isolation and cDNA synthesis For RNA extraction left lung lobes were isolated and snap-froze