Mast Cells and Fibroblasts Work in Concert to Aggravate Pulmonary Fibrosis
2013; Elsevier BV; Volume: 182; Issue: 6 Linguagem: Inglês
10.1016/j.ajpath.2013.02.013
ISSN1525-2191
AutoresMałgorzata Wygrecka, Bhola K. Dahal, Djuro Kosanovic, Frank Petersen, Brigitte Taborski, Susanne von Gerlach, Miroslava Didiášová, Dariusz Zakrzewicz, Klaus T. Preissner, Ralph T. Schermuly, Philipp Markart,
Tópico(s)Asthma and respiratory diseases
ResumoMast cell (MC) accumulation has been demonstrated in the lungs of idiopathic pulmonary fibrosis (IPF) patients. Mediators released from MCs may regulate tissue remodeling processes, thereby contributing to IPF pathogenesis. We investigated the role of MC–fibroblast interaction in the progression of lung fibrosis. Increased numbers of activated MCs, in close proximity to fibroblast foci and alveolar type II cells, were observed in IPF lungs. Correspondingly elevated tryptase levels were detected in IPF lung tissue samples. Coculture of human lung MCs with human lung fibroblasts (HLFs) induced MC activation, as evinced by tryptase release, and stimulated HLF proliferation; IPF HLFs exhibited a significantly higher growth rate, compared with control. Tryptase stimulated HLF growth in a PAR-2/PKC-α/Raf-1/p44/42–dependent manner and potentiated extracellular matrix production, but independent of PKC-α, Raf-1, and p44/42 activities. Proproliferative properties of tryptase were attenuated by knockdown or pharmacological inhibition of PAR-2, PKC-α, Raf-1, or p44/42. Expression of transmembrane SCF, but not soluble SCF, was elevated in IPF lung tissue and in fibroblasts isolated from IPF lungs. Coculture of IPF HLFs with MCs enhanced MC survival and proliferation. These effects were cell-contact dependent and could be inhibited by application of anti-SCF antibody or CD117 inhibitor. Thus, fibroblasts and MCs appear to work in concert to perpetuate fibrotic processes and so contribute to lung fibrosis progression. Mast cell (MC) accumulation has been demonstrated in the lungs of idiopathic pulmonary fibrosis (IPF) patients. Mediators released from MCs may regulate tissue remodeling processes, thereby contributing to IPF pathogenesis. We investigated the role of MC–fibroblast interaction in the progression of lung fibrosis. Increased numbers of activated MCs, in close proximity to fibroblast foci and alveolar type II cells, were observed in IPF lungs. Correspondingly elevated tryptase levels were detected in IPF lung tissue samples. Coculture of human lung MCs with human lung fibroblasts (HLFs) induced MC activation, as evinced by tryptase release, and stimulated HLF proliferation; IPF HLFs exhibited a significantly higher growth rate, compared with control. Tryptase stimulated HLF growth in a PAR-2/PKC-α/Raf-1/p44/42–dependent manner and potentiated extracellular matrix production, but independent of PKC-α, Raf-1, and p44/42 activities. Proproliferative properties of tryptase were attenuated by knockdown or pharmacological inhibition of PAR-2, PKC-α, Raf-1, or p44/42. Expression of transmembrane SCF, but not soluble SCF, was elevated in IPF lung tissue and in fibroblasts isolated from IPF lungs. Coculture of IPF HLFs with MCs enhanced MC survival and proliferation. These effects were cell-contact dependent and could be inhibited by application of anti-SCF antibody or CD117 inhibitor. Thus, fibroblasts and MCs appear to work in concert to perpetuate fibrotic processes and so contribute to lung fibrosis progression. Mast cells (MCs) originate from CD34-expressing hematopoietic stem cells in the bone marrow. They circulate in the blood as monocyte-like precursors and then home to tissues, where they mature under the influence of stem cell factor (SCF) and local cytokines.1Brown J.M. Wilson T.M. Metcalfe D.D. The mast cell and allergic diseases: role in pathogenesis and implications for therapy.Clin Exp Allergy. 2008; 38: 4-18PubMed Google Scholar MCs are predominantly localized at sites that have direct contact with the external environment, such as the skin, airways, and intestine, where they function as sentinel cells in host defense.2Marshall J.S. Mast-cell responses to pathogens.Nat Rev Immunol. 2004; 4: 787-799Crossref PubMed Scopus (678) Google Scholar Upon activation, MCs release their granule contents, which include proteases (tryptase, chymase, carboxypeptidase), vasoactive amines (histamine, serotonin), proteoglycans (heparin, chondroitin sulfate), and growth factors [transforming growth factor-β1 (TGF-β1), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF)].3Metcalfe D.D. Baram D. Mekori Y.A. Mast cells.Physiol Rev. 1997; 77: 1033-1079Crossref PubMed Scopus (1788) Google Scholar In addition, activated MCs are able to produce a variety of cytokines [IL-1α, IL-6, granulocyte-monocyte colony-stimulating factor (GM-CSF), interferon-α (IFN-α)], chemokines (CCL-2, CCL-5, CXCL-1), and lipid mediators [leukotriene C4 (LTC4), LTB4, prostaglandin 2 (PGE2)].3Metcalfe D.D. Baram D. Mekori Y.A. Mast cells.Physiol Rev. 1997; 77: 1033-1079Crossref PubMed Scopus (1788) Google Scholar The broad spectrum of molecules produced by MCs might explain their varied functions: the recruitment, activation, and differentiation of inflammatory cells4Malaviya R. Abraham S.N. Role of mast cell leukotrienes in neutrophil recruitment and bacterial clearance in infectious peritonitis.J Leukoc Biol. 2000; 67: 841-846PubMed Google Scholar, 5Tani K. Ogushi F. Kido H. Kawano T. Kunori Y. Kamimura T. Cui P. Sone S. Chymase is a potent chemoattractant for human monocytes and neutrophils.J Leukoc Biol. 2000; 67: 585-589PubMed Google Scholar, 6Rajakulasingam K. Hamid Q. O'Brien F. Shotman E. Jose P.J. Williams T.J. Jacobson M. Barkans J. Durham S.R. RANTES in human allergen-induced rhinitis: cellular source and relation to tissue eosinophilia.Am J Respir Crit Care Med. 1997; 155: 696-703Crossref PubMed Scopus (53) Google Scholar and the regulation of vascular permeability,2Marshall J.S. Mast-cell responses to pathogens.Nat Rev Immunol. 2004; 4: 787-799Crossref PubMed Scopus (678) Google Scholar smooth-muscle cell contractility,7Aceves S.S. Chen D. Newbury R.O. Dohil R. Bastian J.F. Broide D.H. Mast cells infiltrate the esophageal smooth muscle in patients with eosinophilic esophagitis, express TGF-beta1, and increase esophageal smooth muscle contraction.J Allergy Clin Immunol. 2010; 126: 1198-1204.e4Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar and fibroblast growth.8Levi-Schaffer F. Rubinchik E. Activated mast cells are fibrogenic for 3T3 fibroblasts.J Invest Dermatol. 1995; 104: 999-1003Crossref PubMed Scopus (53) Google Scholar Thus, MCs have been found to be involved in the pathogenesis of allergic, chronic inflammatory, and fibrotic diseases. An association between MC infiltration and the degree of fibrosis has been found in various kidney disorders, including focal segmental glomerulosclerosis, IgA nephropathy, renal amyloidosis, and lupus nephritis,9Tóth T. Tóth-Jakatics R. Jimi S. Takebayashi S. Increased density of interstitial mast cells in amyloid A renal amyloidosis.Mod Pathol. 2000; 13: 1020-1028Crossref PubMed Scopus (33) Google Scholar, 10Kondo S. Kagami S. Kido H. Strutz F. Müller G.A. Kuroda Y. Role of mast cell tryptase in renal interstitial fibrosis.J Am Soc Nephrol. 2001; 12: 1668-1676PubMed Google Scholar, 11Hiromura K. Kurosawa M. Yano S. Naruse T. Tubulointerstitial mast cell infiltration in glomerulonephritis.Am J Kidney Dis. 1998; 32: 593-599Abstract Full Text PDF PubMed Scopus (88) Google Scholar as well as in liver cirrhosis12Yamashiro M. Kouda W. Kono N. Tsuneyama K. Matsui O. Nakanuma Y. Distribution of intrahepatic mast cells in various hepatobiliary disorders. An immunohistochemical study.Virchows Arch. 1998; 433: 471-479Crossref PubMed Scopus (52) Google Scholar and in the minor salivary glands of patients with Sjögren's syndrome.13Skopouli F.N. Li L. Boumba D. Stefanaki S. Hanel K. Moutsopoulos H.M. Krilis S.A. Association of mast cells with fibrosis and fatty infiltration in the minor salivary glands of patients with Sjögren's syndrome.Clin Exp Rheumatol. 1998; 16: 63-65PubMed Google Scholar In addition, a growing body of evidence suggests that MCs may play a role in the pathogenesis of fibrotic lung diseases. Elevated numbers of MCs were found in the lungs of patients with sarcoidosis,14Ohrn M.B. Sköld C.M. van Hage-Hamsten M. Sigurdardottir O. Zetterström O. Eklund A. Sarcoidosis patients have bronchial hyperreactivity and signs of mast cell activation in their bronchoalveolar lavage.Respiration. 1995; 62: 136-142Crossref PubMed Scopus (29) Google Scholar cryptogenic organizing pneumonia,15Schildge J. Klar B. Hardung-Backes M. Die Mastzelle in der bronchoalveolären Lavage bei interstitiellen Lungenerkrankungen.Pneumologie. 2003; 57 (German): 202-207Crossref PubMed Scopus (14) Google Scholar hypersensitivity pneumonitis,15Schildge J. Klar B. Hardung-Backes M. Die Mastzelle in der bronchoalveolären Lavage bei interstitiellen Lungenerkrankungen.Pneumologie. 2003; 57 (German): 202-207Crossref PubMed Scopus (14) Google Scholar silicosis,16Hamada H. Vallyathan V. Cool C.D. Barker E. Inoue Y. Newman L.S. Mast cell basic fibroblast growth factor in silicosis.Am J Respir Crit Care Med. 2000; 161: 2026-2034Crossref PubMed Scopus (30) Google Scholar and idiopathic pulmonary fibrosis (IPF).17Hunt L.W. Colby T.V. Weiler D.A. Sur S. Butterfield J.H. Immunofluorescent staining for mast cells in idiopathic pulmonary fibrosis: quantification and evidence for extracellular release of mast cell tryptase.Mayo Clin Proc. 1992; 67: 941-948Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 18Cha S.I. Chang C.S. Kim E.K. Lee J.W. Matthay M.A. Golden J.A. Elicker B.M. Jones K. Collard H.R. Wolters P.J. Lung mast cell density defines a subpopulation of patients with idiopathic pulmonary fibrosis.Histopathology. 2012; 61: 98-106Crossref PubMed Scopus (46) Google Scholar, 19Andersson C.K. Andersson-Sjöland A. Mori M. Hallgren O. Pardo A. Eriksson L. Bjermer L. Löfdahl C.G. Selman M. Westergren-Thorsson G. Erjefält J.S. Activated MCTC mast cells infiltrate diseased lung areas in cystic fibrosis and idiopathic pulmonary fibrosis.Respir Res. 2012; 12: 139Crossref Scopus (59) Google Scholar Moreover, increased levels of tryptase were measured in bronchoalveolar lavage fluid (BALF) from IPF patients, and tryptase-positive IPF cases were reported to have a poorer outcome.20Kawatani K. Kondo M. Tamaoki J. Tagaya E. Nagai A. Nihon Kokyuki Gakkai Zasshi. 2007; 45 (Japanese): 848-855PubMed Google Scholar The possible involvement of MCs in the pathogenesis of IPF may arise from their ability to produce a variety of profibrotic factors. In this regard, tryptase, the most abundant protein in human lung MC, was found to stimulate collagen I synthesis21McLarty J.L. Meléndez G.C. Brower G.L. Janicki J.S. Levick S.P. Tryptase/Protease-activated receptor 2 interactions induce selective mitogen-activated protein kinase signaling and collagen synthesis by cardiac fibroblasts.Hypertension. 2011; 58: 264-270Crossref PubMed Scopus (70) Google Scholar and fibroblast proliferation.8Levi-Schaffer F. Rubinchik E. Activated mast cells are fibrogenic for 3T3 fibroblasts.J Invest Dermatol. 1995; 104: 999-1003Crossref PubMed Scopus (53) Google Scholar Similar cellular activities were reported on exposure of fibroblasts to TGF-β1, bFGF, or histamine,22Hetzel M. Bachem M. Anders D. Trischler G. Faehling M. Different effects of growth factors on proliferation and matrix production of normal and fibrotic human lung fibroblasts.Lung. 2005; 183: 225-237Crossref PubMed Scopus (165) Google Scholar which are other mediators released from activated MC. Moreover, it was shown that both tryptase and chymase are capable of activating matrix metalloproteinases, thereby contributing to extracellular matrix (ECM) turnover.23Tchougounova E. Lundequist A. Fajardo I. Winberg J.O. Abrink M. Pejler G. A key role for mast cell chymase in the activation of pro-matrix metalloprotease-9 and pro-matrix metalloprotease-2.J Biol Chem. 2005; 280: 9291-9296Crossref PubMed Scopus (276) Google Scholar, 24Iddamalgoda A. Le Q.T. Ito K. Tanaka K. Kojima H. Kido H. Mast cell tryptase and photoaging: possible involvement in the degradation of extra cellular matrix and basement membrane proteins.Arch Dermatol Res. 2008; 300: S69-S76Crossref PubMed Scopus (78) Google Scholar Although MC mediators are known to have profibrotic properties, the molecular mechanism of their action is poorly understood. The major focus of the present study was therefore to decipher the molecular basis of MC–fibroblast interplay in the development of pulmonary fibrosis. The investigations were conducted according to Declaration of Helsinki principles and were approved by the local institutional ethics committee. Informed consent was obtained from either the patients or their next of kin. BALF was obtained by flexible fiberoptic bronchoscopy from 20 spontaneously breathing healthy volunteers and from 40 spontaneously breathing IPF patients. Diagnosis of IPF was decided on the basis of recently published guidelines.25Raghu G. Collard H.R. Egan J.J. Martinez F.J. Behr J. Brown K.K. Colby T.V. Cordier J.F. Flaherty K.R. Lasky J.A. Lynch D.A. Ryu J.H. Swigris J.J. Wells Ancochea J. Bouros D. Carvalho C. Costabel U. Ebina M. Hansell D.M. Johkoh T. Kim D.S. King Jr., T.E. Kondoh Y. Myers J. Müller N.L. Nicholson A.G. Richeldi L. Selman M. Dudden R.F. Griss B.S. Protzko S.L. Schünemann H.J. ATS/ERS/JRS/ALAT Committee on Idiopathic Pulmonary FibrosisAn official ATS/ERS/JRS/ALAT statement: idiopathic pulmonary fibrosis: evidence-based guidelines for diagnosis and management.Am J Respir Crit Care Med. 2011; 183: 788-824Crossref PubMed Scopus (5369) Google Scholar In 20 patients, diagnosis was confirmed by surgical lung biopsy, which revealed a pattern of usual interstitial pneumonia in every case. Additionally, lung tissue was obtained from 24 IPF patients who underwent lung transplantation at the Department of Cardiothoracic Surgery, Medical University of Vienna, Austria. IPF diagnosis was based on clinical criteria, as well as proof of a usual interstitial pneumonia pattern. Nonused donor lungs served as control (n = 10). Inflammatory processes were not observed in donor lungs by histopathological evaluation. Demographic and clinical characteristics of the patient cohort are reported elsewhere.26Wygrecka M. Kwapiszewska G. Jablonska E. von Gerlach S. Henneke I. Zakrzewicz D. Guenther A. Preissner K.T. Markart P. Role of protease-activated receptor-2 in idiopathic pulmonary fibrosis.Am J Respir Crit Care Med. 2011; 183: 1703-1714Crossref PubMed Scopus (65) Google Scholar For isolation of human lung MCs, tumor-free lung tissue obtained from patients with bronchial carcinoma undergoing lobectomy was provided by the Section of Pathology, Research Center Borstel. Approval for these studies was obtained from the institutional review board at the University of Lübeck, and informed consent was provided according to the Declaration of Helsinki. Lung specimens were chopped into pieces and placed overnight in ice-cold MC buffer (12 mmol/L HEPES, pH 7.4, 290 mmol/L NaCl, 3 mmol/L KCl, 3.7 mmol/L Na3PO4·12H2O, 6 mmol/L glucose, 0.1% bovine serum albumin). To disperse cells enzymatically, lung pieces were incubated in MC buffer containing 0.1% gelatin, 1.5 mg/mL dispase II, 0.375 mg/mL chymopapain (both from Sigma-Aldrich, Taufkirchen, Germany; St. Louis, MO), 0.75 mg/mL collagenase type I, 1.79 mg/mL elastase (both from Worthington, Lakewood, NJ) under agitation for 4 hours at 37°C. MCs were enriched by Percoll gradient centrifugation followed by immunoaffinity magnetic enrichment, using anti-phycoerythrin–conjugated microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany; Auburn, CA) in combination with phycoerythrin-conjugated mAb 97A6 (IOTest; Beckman Coulter–Immunotech, Marseille, France; Fullerton, CA) specific for CD203c. The purity of MCs used for experiments ranged from 88% to 100% as assessed by Toluidine Blue staining. Viability was always >85% as assessed by Trypan Blue exclusion. Purified MCs (5 × 105/mL) were cultured in StemPro medium (Life Technologies–Invitrogen, Carlsbad, CA) containing 2 mmol/L l-glutamine, 50 IU/mL penicillin, 50 μg/mL streptomycin, and 100 ng/mL human recombinant SCF (PeproTech, London, UK; Rocky Hill, NJ). MCs taken from culture 5 days after isolation were washed with fetal calf serum–free medium and were seeded onto confluent serum-starved human lung fibroblasts (HLFs) at a 1:2 ratio. When required, 100 nmol/L of the CD117 inhibitor ISCK03 (Santa Cruz Biotechnology, Santa Cruz, CA), 10 μg/mL anti-human SCF (R&D Systems, Wiesbaden, Germany; Minneapolis, MN), or 10 μg/mL isotype IgG control (R&D Systems) was included. MC numbers during the coculture period were assessed using Kimura staining, which readily differentiates red metachromatic MCs from unlabeled HLFs. MC monoculture controls were established in parallel. To investigate tryptase release, MCs were incubated with control or IPF HLFs in Dulbecco's modified Eagle's medium alone or in the presence of 2 μg/mL compound 48/80 (C48/80; Sigma-Aldrich), or were challenged with BALF obtained from healthy control subjects or IPF patients. After 4 hours, cells and supernatants were separated by centrifugation and supernatants were analyzed for the presence of tryptase by Western blotting. HLFs were isolated from control (donor) and IPF lungs, as described previously.27Jablonska E. Markart P. Zakrzewicz D. Preissner K.T. Wygrecka M. Transforming growth factor-beta1 induces expression of human coagulation factor XII via Smad3 and JNK signaling pathways in human lung fibroblasts.J Biol Chem. 2010; 285: 11638-11651Crossref PubMed Scopus (38) Google Scholar HLFs were either not stimulated or were stimulated for various time periods with different concentrations of tryptase (0.5 to 4 nmol/L; R&D Systems). In some experiments, cells were transfected with 100 nmol/L human proteinase activated receptor 2 (PAR-2) siRNA (Santa Cruz Biotechnology), human PKC-α siRNA (Ambion; Life Technologies, Austin, TX), or universal negative control siRNA (Ambion; Life Technologies) using siLentFect lipid reagent (Bio-Rad Laboratories, Munich, Germany; Hercules, CA) 48 hours before the addition of 2 nmol/L tryptase. In other experiments, cells were pretreated with the PAR-2 antagonist ENMD-1068 (Enzo Life Sciences, Lörrach, Germany; Farmingdale, NY), Gö 6976, Ro 32-0432, Raf-1 inhibitor, or PD 98059 (all from Calbiochem; EMD-Millipore, Darmstadt, Germany; Billerica, MA) 1 hour before exposure to 2 nmol/L tryptase. Proliferation of HLFs was determined by a DNA synthesis assay based on the uptake of [3H]thymidine (Amersham; GE Healthcare, Freiburg, Germany; Little Chalfont, UK). Cells were cultured in 48-well plates, growth-arrested in serum-free Dulbecco's modified Eagle's medium, and left unstimulated or stimulated for 24 hours in serum-free medium with 0.5 to 4 nmol/L tryptase, 65 U/L thrombin (American Diagnostica; Sekisui Diagnostics, Lexington, MA), 100 μmol/L PAR-2 agonist peptide (2-furoyl-LIGRLO-NH2), or 100 μmol/L scrambled peptide control (trans-cinnamoyl-OLIGRL-NH2) (both of the latter kindly provided by Dr. A. Meinhardt, Justus-Liebig-University, Giessen, Germany). Subsequently, the cells were pulsed with 1.2 μCi/mL [3H]thymidine for 16 hours. Afterward, cells were solubilized in 0.5 mol/L NaOH, and [3H]thymidine incorporation was determined by liquid scintillation spectrometry. In some experiments, ENMD-1068, Gö 6976, Ro 32-0432, Raf-1 inhibitor, or PD 98059 (Calbiochem; EMD-Millipore) was added to the cells 1 hour before stimulation. In other experiments, cells were transfected with human PAR-2 siRNA (Santa Cruz Biotechnology), human PKC-α siRNA, or universal negative control siRNA (100 nmol/L each; Ambion; Life Technologies) 48 hours before the proliferation assay. HLFs were seeded at a density of 10,000 cells per well in 48-well plates. Cocultures were initiated by introducing human lung MCs to HLFs at a 1:2 ratio. Cocultures were maintained in Dulbecco's modified Eagle's medium alone or with additives [2 μg/mL C48/80 (Sigma-Aldrich) and APC366 (R&D Systems)] for various time periods. In some experiments, HLFs were pretreated with 50 μg/mL of the PAR-2 antagonist ENMD-1068 (Calbiochem; EMD-Millipore) 1 hour before incubation with MCs. In other experiments, PAR-2 was depleted in HLFs 48 hours before incubation with MCs. At the end of the coculture experiments, MCs were washed away and HLFs were subjected to measurement of [3H]thymidine incorporation. SCF antigen level was determined in BALF samples of IPF patients and healthy control subjects using a human SCF enzyme-linked immunosorbent assay kit (R&D Systems) according to the manufacturer's instructions. RNA isolation and quantitative real-time RT-PCR (RT-qPCR) were performed as described previously.28Wygrecka M. Wilhelm J. Jablonska E. Zakrzewicz D. Preissner K.T. Seeger W. Guenther A. Markart P. Shedding of low-density lipoprotein receptor-related protein-1 in acute respiratory distress syndrome.Am J Respir Crit Care Med. 2011; 184: 438-448Crossref PubMed Scopus (31) Google Scholar The following oligonucleotide primers were used: human soluble stem cell factor (SCFS) forward, 5′-GGACTTTGTAGTGGCATCTGAA-3′, human SCFS reverse, 5′-CTAAGGGAGCTGGCTGCAA-3′; human transmembrane SCF (SCFTM) forward, 5′-CCTGAGAAAGGGAAGGCCAA-3′, human SCFTM reverse, 5′-CTTATACTGGAAGAAGAGA-3′; human fibronectin (FN) forward, 5′-TGTACTTCTGAGGGTCGCAGG-3′, human FN reverse, 5′-CCAATGCGATACATGACCCC-3′; human collagen I forward, 5′-CAAGAGGAAGGCCAAGTCGAG-3′; human collagen I reverse; 5′-TTGTCGCAGACGCAGATCC-3′; human α-SMA forward, 5′-CGAGATCTCACTGACTACCTCATGA-3′; human α-SMA reverse, 5′-AGAGCTACATAACACAGTTTCTCCTTGA-3; human β-actin forward: 5′-ATTGCCGACAGGATGCAGGAA-3′, human β-actin reverse: 5′-GCTGATCCACATCTGCTGGAA-3′. β-actin was used as the reference gene. Cycling conditions were 95°C for 6 minutes, followed by 40 cycles of 95°C for 20 seconds, 58°C for 30 seconds, and 73°C for 30 seconds. Melting-curve analysis and gel electrophoresis were performed to confirm the exclusive amplification of the expected PCR product. Lung tissue samples or cells were lysed in ice-cold lysis buffer [20 mmol/L Tris pH 7.5, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton X-100, 2.5 μmol/L Na-pyrophosphate, 1 mmol/L β-glycerophosphate, 1 mmol/L Na3VO4, 1 mmol/L phenylmethylsulfonyl fluoride, 1 μg/mL Complete Protease Inhibitor Cocktail (Roche Applied Science, Indianapolis, IN)]. Protein lysates were separated on a 10% SDS polyacrylamide gel under reducing conditions, followed by electrotransfer to a polyvinylidene difluoride membrane. After blocking, the membranes were probed with one of the following antibodies: mouse anti-MC tryptase (R&D Systems), rabbit anti-SCF, mouse anti-FN, rabbit anti-histone H2B (all from Abcam, Cambridge, MA), mouse anti–phospho-p44/42, rabbit anti–phospho-PKC(pan), rabbit anti–phospho-PKC-δ (Thr 505), rabbit anti–phospho-Raf-1 (all from Cell Signaling Technology, Danvers, MA), rabbit anti-EGFR, rabbit anti–phospho-PKC-α (Thr 638; Biomol; Enzo Life Sciences, Hamburg, Germany; Plymouth Meeting, PA), or rabbit anti–phospho-PKC-ε (Ser 729; Life Technologies, Darmstadt, Germany; Carlsbad, CA). Membranes were then incubated with peroxidase-labeled secondary antibodies (all from Dako, Glostrup, Denmark). Final detection of proteins was performed using an ECL Plus kit (Amersham; GE Healthcare, Freiburg, Germany). To determine the amounts of protein loaded on the gel, blots were stripped and reprobed using mouse anti–β-actin (Sigma-Aldrich), rabbit anti-p44/42, rabbit anti–Raf-1, rabbit anti–PKC-α, rabbit anti–PKC-ε, or rabbit anti–PKC-δ (all from Cell Signaling Technology). Under a standard protocol, paraffin-embedded lung tissue sections (3 μm thick) from IPF patients and control subjects were stained with Toluidine Blue to identify MCs. The tissue sections were dewaxed, rehydrated, and incubated with 0.05% (w/v) Toluidine Blue for 2 to 3 minutes. The number of MCs in each section was counted, and MCs were identified as granulated or degranulated. The index of degranulation, which is calculated as the number of degranulated MCs divided by the number of granulated MCs, was determined as described previously29Dahal B.K. Kosanovic D. Kaulen C. Cornitescu T. Savai R. Hoffmann J. Reiss I. Ghofrani H.A. Weissmann N. Kuebler W.M. Seeger W. Grimminger F. Schermuly R.T. Involvement of mast cells in monocrotaline-induced pulmonary hypertension in rats.Respir Res. 2011; 12: 60Crossref PubMed Scopus (62) Google Scholar and was expressed as a percentage, assuming 100% for control subjects. The MC analysis was done by investigators masked to patient versus control group (B.K.D. and D.K.). Paraffin-embedded lung tissue sections (5 μm thick) from IPF patients and control subjects were deparaffinized in xylene and rehydrated through graded ethanol washes. Antigen retrieval was performed by cooking tissue sections for 30 minutes in Tris-EDTA buffer (10 mmol/L Tris, pH 9.0, 1 mmol/L EDTA). Immunohistochemistry was performed using a ZytoChem-Plus AP polymer kit according to the manufacturer's instructions (Zymed Laboratories, San Francisco, CA). The following primary antibodies were used: mouse anti-MC tryptase, rabbit anti-SCF (both from Abcam), or mouse anti–α-SMA (Sigma-Aldrich). For negative control, the primary antibody was replaced with a species-matched isotype control. In the illustrations, images are representative of at least six other areas per section, seen on at least three independent sections per patient. Tryptase-positive MCs in each section were counted using a light microscope. The mean number of tryptase-positive MCs was expressed as a percentage, assuming 100% in control subjects. The MC analysis was done by investigators masked to patient versus control group (B.K.D. and D.K.). For immunocytochemical analysis, HLFs were stimulated with 2 nmol/L tryptase for 30 minutes. After a PBS wash, the cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.2% Triton X-100 in PBS for 10 minutes, blocked with 3% bovine serum albumin in PBS for 1 hour at room temperature, and incubated overnight at 4°C with rabbit anti–PKC-α antibody (Biomol; Enzo Life Sciences). Slides were then incubated with Alexa Fluor 568–conjugated secondary antibody (Dianova, Hamburg, Germany) and mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA). Nuclei were visualized by DAPI (Sigma-Aldrich) staining. For negative control, the primary antibody was replaced by a species-matched isotype control. Images were captured under a Leica DMR microscope (Leica Microsystems, Wetzlar, Germany) with a 40× objective. Statistical analyses of in vitro data were performed using one-way analysis of variance followed by Tukey's honestly significant difference post hoc test. Clinical data are expressed as median and interquartile range; box-and-whisker-plots indicate the median, first, and third quartile, with the whiskers extended to the most extreme value inside the 1.5-fold interquartile range. Statistical analyses of clinical data were performed using Wilcoxon rank sum test. The level of statistical significance was set at P ≤ 0.05. Previous studies have demonstrated increased numbers of MCs in lungs of IPF patients.17Hunt L.W. Colby T.V. Weiler D.A. Sur S. Butterfield J.H. Immunofluorescent staining for mast cells in idiopathic pulmonary fibrosis: quantification and evidence for extracellular release of mast cell tryptase.Mayo Clin Proc. 1992; 67: 941-948Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 18Cha S.I. Chang C.S. Kim E.K. Lee J.W. Matthay M.A. Golden J.A. Elicker B.M. Jones K. Collard H.R. Wolters P.J. Lung mast cell density defines a subpopulation of patients with idiopathic pulmonary fibrosis.Histopathology. 2012; 61: 98-106Crossref PubMed Scopus (46) Google Scholar, 19Andersson C.K. Andersson-Sjöland A. Mori M. Hallgren O. Pardo A. Eriksson L. Bjermer L. Löfdahl C.G. Selman M. Westergren-Thorsson G. Erjefält J.S. Activated MCTC mast cells infiltrate diseased lung areas in cystic fibrosis and idiopathic pulmonary fibrosis.Respir Res. 2012; 12: 139Crossref Scopus (59) Google Scholar In accord with these reports, we also observed elevated number of MCs in IPF lungs (Figure 1A ); moreover, the majority of MCs in IPF lungs were activated, as evinced by an increased index of degranulation (Figure 1B). Because tryptase-positive MCs are the dominant MC type in human peripheral lung, we subsequently evaluated the number of tryptase-positive cells in the lungs of IPF patients and control subjects. Staining of serial IPF lung tissue sections with Toluidine Blue and for tryptase demonstrated that Toluidine Blue–stained MCs are also tryptase-positive (Figure 1C). Consistently, the number of tryptase-positive cells was elevated in the lungs of IPF patients, compared with control subjects (Figure 1D). Tryptase-positive cells were located in close proximity to fibroblast foci and hyperplastic alveolar type II cells (Figure 1E). Moreover, Western blot analysis revealed increased tryptase levels in IPF lung homogenates (Figure 1, F and G). To assess the role of MCs in the proliferation of HLFs, we performed coculture experiments with isolated human lung MCs and HLFs from IPF patients and control subjects. Coincubation of MCs with HLFs induced HLF proliferation (Figure 2A ). Notably, IPF fibroblasts displayed a significantly more enhanced growth rate, compared with control fibroblasts (Figure 2A). Furthermore, coculture of HLFs with MCs induced MC degranulation, as evinced by the presence of tryptase in cell culture medium (Figure 2B). These effects were enhanced when MCs were additionally activated by C48/80 (Figure 2, A and B). Because no major difference in the extent of MC activation between donor and IPF HLFs was observed, we considered that factors produced by other cell types might contribute to the elevated number of degranulated MCs observed in IPF lungs, compared with control subjects. Consistent with this notion, potentiated degranulation of MCs was observed o
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