Lipopolysaccharide-Activated Macrophages Stimulate the Synthesis of Collagen Type I and C-Fibronectin in Cultured Pancreatic Stellate Cells
1999; Elsevier BV; Volume: 155; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)65490-9
ISSN1525-2191
AutoresAlexandra Schmid‐Kotsas, Hans‐Jürgen Groß, André Menke, H Weidenbach, Guido Adler, Marco Siech, Hans G. Beger, A. Grünert, Max G. Bachem,
Tópico(s)Pediatric Hepatobiliary Diseases and Treatments
ResumoWe have recently identified and characterized pancreatic stellate cells (PSC) in rats and humans (Gastroenterology 1998, 15:421–435). PSC are suggested to represent the main cellular source of extracellular matrix in chronic pancreatitis. Now we describe a paracrine stimulatory loop between human macrophages and PSC (rat and human) that results in an increased extracellular matrix synthesis. Native and transiently acidified supernatants of cultured macrophages were added to cultured PSC in the presence of 0.1% fetal calf serum. Native supernatants of lipopolysaccharide-activated macrophages stimulated the synthesis of collagen type I 1.38 ± 0.09-fold of control and c-fibronectin 1.89 ± 0.18-fold of control. Transiently acidified supernatants stimulated collagen type I and c-fibronectin 2.10 ± 0.2-fold and 2.80 ± 0.05-fold of control, respectively. Northern blot demonstrated an increased expression of the collagen-I-(α-1)-mRNA and fibronectin-mRNA in PSC 10 hours after addition of the acidified macrophage supernatants. Cell proliferation measured by bromodeoxyuridine incorporation was not influenced by the macrophage supernatants. Unstimulated macrophages released 1.97 pg TGFβ1/μg of DNA over 24 hours and lipopolysaccharide-activated macrophages released 6.61pg TGFβ1/μg of DNA over 24 hours. These data together with the results that, in particular, transiently acidified macrophage supernatants increased matrix synthesis, identify TGFβ as the responsible mediator. In conclusion, our data demonstrate a paracrine stimulation of matrix synthesis of pancreatic stellate cells via TGFβ1 released by activated macrophages. We suggest that macrophages might play a pivotal role in the development of pancreas fibrosis. We have recently identified and characterized pancreatic stellate cells (PSC) in rats and humans (Gastroenterology 1998, 15:421–435). PSC are suggested to represent the main cellular source of extracellular matrix in chronic pancreatitis. Now we describe a paracrine stimulatory loop between human macrophages and PSC (rat and human) that results in an increased extracellular matrix synthesis. Native and transiently acidified supernatants of cultured macrophages were added to cultured PSC in the presence of 0.1% fetal calf serum. Native supernatants of lipopolysaccharide-activated macrophages stimulated the synthesis of collagen type I 1.38 ± 0.09-fold of control and c-fibronectin 1.89 ± 0.18-fold of control. Transiently acidified supernatants stimulated collagen type I and c-fibronectin 2.10 ± 0.2-fold and 2.80 ± 0.05-fold of control, respectively. Northern blot demonstrated an increased expression of the collagen-I-(α-1)-mRNA and fibronectin-mRNA in PSC 10 hours after addition of the acidified macrophage supernatants. Cell proliferation measured by bromodeoxyuridine incorporation was not influenced by the macrophage supernatants. Unstimulated macrophages released 1.97 pg TGFβ1/μg of DNA over 24 hours and lipopolysaccharide-activated macrophages released 6.61pg TGFβ1/μg of DNA over 24 hours. These data together with the results that, in particular, transiently acidified macrophage supernatants increased matrix synthesis, identify TGFβ as the responsible mediator. In conclusion, our data demonstrate a paracrine stimulation of matrix synthesis of pancreatic stellate cells via TGFβ1 released by activated macrophages. We suggest that macrophages might play a pivotal role in the development of pancreas fibrosis. 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The presence of both pro-TGFβ and LTBP in monocytes and/or macrophages in areas of fibrosis strongly suggests that this cytokine is involved in the pathophysiology of chronic pancreatitis.27Van Laethem JL Deviere J Resibois A Rickaert F Vertongen P Ohtani H Cremer M Miyazono K Robberecht P Localization of transforming growth factor β1 and its latent binding protein in human chronic pancreatitis.Gastroenterology. 1995; 108: 1873-1881Abstract Full Text PDF PubMed Scopus (145) Google Scholar In the present study, we demonstrate that transiently acidified supernatants of LPS stimulated human monocyte-derived macrophages contain TGFβ and stimulated the synthesis of fibronectin and collagen type I in cultured PSCs. These findings suggest that activated macrophages play a critical role in pancreas fibrogenesis by stimulating matrix synthesis of PSCs in a paracrine way. Materials were purchased from the following sources: Ficoll-Paque from Pharmacia, Biotech (Uppsala, Sweden); biotin labeled CD14(My4) and CD3-ECD from Coulter Immunotech (Hamburg, Germany); rabbit anti-human collagen type I and biotin-labeled goat anti-human collagen type III from Chemicon (Temecula, CA); rabbit anti-fibronectin from Behring Diagnostics (Marburg, Germany); biotin labeled anti-rabbit, biotin labeled anti-mouse, HRP anti-rabbit, biotin labeled anti-goat, HRP anti-mouse, fluorescein-conjugated streptavidin, HRP-conjugated streptavidin, mouse anti-BrdU, anti-CD19-PECy5, and anti-CD14-PE from DAKO (Hamburg, Germany); streptavidin-Red 613 from Gibco BRL (Eggenstein, Germany); mouse anti-α-smooth-muscle actin, bromodeoxyuridine, and the High Pure RNA Extraction Kit from Boehringer Mannheim (Mannheim, Germany); TSA Indirect from NEN Life Science Products (Boston, MA); fluorescein-conjugated Escherichia coli and propidium jodide (Orpegen, Heidelberg, Germany); trypan blue, bisbenzimide, calf thymus DNA, yeast t-RNA, diethylendtriaminepentaacetic (DTPA), monoclonal anti-fibronectin, and β-aminopropionitrile from Sigma (Deisenhofen, Germany); ascorbic acid from Merck (Darmstadt, Germany); enhancement solution and europium-conjugated streptavidin from Wallac Oy (Turku, Finland); and TGFβ1-sR-II/Fc chimera and biotinylated anti-human TGFβ1 from R&D Systems (Minneapolis, MN). The 18S rRNA probe was generously provided by Dr. T. M. Gress (University Ulm, Germany). Ninety-six-well microtiter plates (Maxi Sorp) were from Nunc GmbH (Wiesbaden, Germany), cell culture plates and flasks were from Falcon (Becton Dickinson, Heidelberg, Germany) and petriPerm was from In Vitro Systems + Services (Osterode, Germany). Hybond-N membranes were purchased from Amersham-Buchler (Braunschweig, Germany). Peripheral blood mononuclear cells were isolated from buffy coats of different donors by Ficoll-Paque gradient centrifugation. The mononuclear fraction was washed in PBS and then resuspended in RPMI 1640 containing 1% L-glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin and 10% fetal calf serum (FCS). Aliquots of the cell suspension (5 × 106 mononuclear cells/ml. were allowed to adhere in 75 cm2 tissue culture flasks in 37°C in a humidified 5% CO2 environment. For immunofluorescence staining cells were seeded on glass coverslips (1 cm2) in 6-well plates. To harvest cells for flow cytometric analysis, cells were seeded on petriPerm. After 2 hours the nonadherent cells were removed and fresh medium was added. Medium was changed each third day and cells were cultured for up to 14 days. Cultured macrophages in 75-cm2 flasks (10–14 days after seeding) were washed with RPMI 1640. Thereafter 10 ml fresh RPMI per flask (with antibiotics, without FCS) was added and conditioned for 24 hours in the absence and presence of 8 μg/ml LPS. Conditioned media were removed under sterile conditions, centrifuged (800 rpm, 5 minutes, 4°C) to remove cell debris, and stored at −80°C. The media were dialyzed for 36 hours against 100 vol distilled water at 4°C in tubings (ZelluTrans, Roth, Karlsruhe, Germany) with 3.5-kd molecular cutoff. Thereafter media were concentrated 10-fold by lyophilization and sterilized by passing through a 0.22-μm pore size filter (Millex-GS, Millipore). To activate latent TGFβ1, aliquots of the media were acidified with 1 mol/L HCl for 10 minutes, then neutralized by adding 1.2 N NaOH/0.5 mol/L Hepes. Human pancreatic stellate cells were isolated by outgrowth, using explant techniques from histologically fibrotic areas of the pancreas surgically resected from patients with chronic pancreatitis. Small tissue blocks were cut (0.5–1 mm3) and seeded in 10-cm2 uncoated culture wells (6 per plate, 3–5 pieces/well) in the presence of 10 to 20% FCS in a 1:1 (v:v) mixture of Dulbecco's modified Eagle's medium (DMEM) with Ham's F12 medium. L-glutamine (2 mmol/L), penicillin/streptomycin, and amphotericine were freshly added. Tissue blocks were cultured at 37°C in a 5. CO2-air humidified atmosphere. Eighteen hours after seeding, culture medium was changed and 24 hours later the small tissue blocks were transferred to new culture plates. The pancreatic stellate cells grew out in high number and purity from the tissue blocks 1 to 3 days later. The small tissue blocks were removed after 2 to 3 weeks. To obtain a higher number of cells with the inactivated resting fat storing phenotype, the cells were isolated by density gradient centrifugation from the pancreas of untreated male Wistar rats as described.11Bachem MG Schneider E Gross H Widenbach H Schmid RM Menke A Siech M Beger H Grünert A Adler G Identification, culture, and characerization of pancreatic stellate cells in rats, and humans.Gastroenterology. 1998; 115: 421-432Abstract Full Text Full Text PDF PubMed Scopus (878) Google Scholar Briefly, after the animals were anesthetized with pentobarbital, the abdomen was opened, the common bile duct was ligated, and a cannula was inserted into the biliopancreatic duct. The rats were exsanguinated and collagenase-containing Eagle's medium (1 mg/5 ml) was instilled intraductally. The distended pancreas was removed and shaken in an Erlenmeyer flask (37°C, 15 minutes.). After this first digestion the pancreas was minced, followed by a second digestion with collagenase (1.75 mg/5 ml, 45 minutes.). Dispersion was accomplished by up-and-down suction through cannulas with decreasing diameters. After dissociation the acini and cells were filtered through a 250-μm nylon cloth and centrifuged after layering the filtrate on top of a dextran-Eagle-HEPES density gradient. Once centrifuged, cells were collected from the top of the gradient, washed twice, resuspended in Tris-buffered saline, and transferred on top of a Iodixanol (OptiPrep) density gradient. After another centrifugation, cells were collected from the top of the gradient, washed, and suspended in DMEM with 10% FCS, antibiotics, amphotericine, and L-glutamine. Thereafter cells were seeded in a density of 4 × 104 cells/cm2. Cells were cultured at 37°C in a 5% CO2 humidified atmosphere. The medium consisted of DMEM/Ham's F12 (1:1, v:v) with 10% FCS, 2% L-glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 1% amphotericine. Medium was changed 3 times a week. After reaching confluency, cells were subcultured by trypsinization using a 0.025% trypsin solution containing 0.01% EDTA in phospate-buffered saline. Once the cell numbers were counted, they were again suspended in a complete medium and seeded with a density of 3–5 × 104 cells/cm2. To increase the cell number to obtain sufficient amounts of RNA, cells were seeded in 75-cm2 culture flasks (25 ml medium/flask). Experiments were performed using PSC between passage 3–8. After passage PSC were cultured for 2 to 3 days in DMEM/HAM's F12 (1/1, v/v. in the presence of 10% FCS. Twelve hours before addition of macrophage supernatants, medium was changed to DMEM/HAM's F12 with 0.1% FCS. To study cell proliferation and matrix synthesis PSC were cultured in 24-well plates (2 cm2/well, 1 ml medium). To study matrix synthesis, native and transiently acidified macrophage supernatants (40 and 160 μl/ml) were added and cultures were stopped 24 hours later. To study cell proliferation BrdU (final concentration 0.5 × 10−5 M) was added 6 hours after addition of 40 μl/ml native and transiently acidified macrophage supernatants. Cultures were stopped 18 hours later. To perform immunofluorescence microscopy of collagen and fibronectin, PSC were seeded on 1-cm2 glass coverslips in 6-well plates and incubated with 40 μl/ml transiently acidified macrophage supernatant. Cultures were stopped 38 hours later. To isolate mRNA, PSCs were grown in 75 cm2 flasks containing 10 ml medium. To subconfluent quiescent cells (cultured for 12 hours in the presence of 0.1% FCS) 40μl/ml transiently acidified macrophage supernatant was added and cultures were stopped 10 hours later. In all experiments the baseline (negative) control represents PSC cultured in DMEM/HAM's F12 with 0.1% FCS. Glass coverslips with cultured hPSC were washed in PBS to remove medium proteins, fixed for 30 minutes in −20°C acetone, and blocked with TNB for 45 minutes. For collagen type I the staining sequence was primary antibody (rabbit-anti-human-collagen I, 1:100), second antibody (HRP-anti-rabbit, 1:100), biotin-TSA-reagent (1:40) and streptavidin-FITC (1:100). For collagen type III the staining sequence was primary antibody (biotin labeled goat-anti-human-collagen III, 1:100), HRP-streptavidin (1:100), biotin-TSA-reagent (1:40) and streptavidin-FITC (1:100). The staining sequence for fibronectin was primary antibody (rabbit-anti-fibronectin, 1:100), second antibody (biotin-anti-rabbit, 1:100) and streptavidin-FITC (1:100). For α-smooth muscle actin the staining sequence was primary antibody (mouse anti-α-smooth muscle actin, 1:50), second antibody (HRP-anti-mouse, 1:50), biotin-TSA-reagent (1:40) and streptavidin-FITC (1:100). Cells were viewed by epifluorescence microscopy (Carl Zeiss, Oberkochen, Germany) with appropriate filter sets. To demonstrate phagocytosis macrophages were incubated with 3 × 107 opsonized FITC-conjugated E. coli for 6 hours at 37°C, washed with PBS, incubated first with biotin-anti-CD14 (1:33) and then with streptavidin-Red 613 (1:100). Thereafter, cells were fixed for 30 minutes in 4% formaldehyde. DNA was counterstained with bisbenzimide. Viability of cultured macrophages was assessed by trypan blue exclusion. To analyze the cells by flow cytometry, macrophages were grown in petriPerm for 10 to 14 days and harvested by scraping. Thereafter cells were washed with PBS, resuspended, and analyzed for expression of CD14, CD3, and CD19 using an EPICS XL flow cytometer (Coulter Immunotech, Hamburg, Germany). Viable cells were detected by propidium jodide exclusion. To measure collagen type I cells were cultured in DMEM/HAM's F12 with 0.1% FCS in the presence of ascorbic acid (100 μg/ml) and β-aminopropionitrile (100 μg/ml). By time-resolved fluorescence-immunoassay collagen type I was measured in culture supernatants 24 hours after stimulation. Briefly, 100 μl cell culture supernatant (diluted 1:4 with 0.05 mol/L NaHCO3, pH 9.1) were transferred to 96-well microtiter plates (Nunc-Maxi Sorp. and incubated overnight at 4°C. After 3 washing steps (wash buffer. Tris 0.05 mol/l, NaCl 0.15 mol/l, Tween 20 0.05%, pH 7.5) plates were blocked during 2 hours with assay buffer (Tris 0.05 mol/l, NaCl 0.15 mol/l, dry milk powder 5%, pH 7.5). Thereafter, the plates were incubated for 3 hours with a polyclonal rabbit-anti-human-collagen type I (diluted 1:500 in assay buffer). After washing 3 times the plates were incubated for 2 hours with the second antibody (biotin-labeled anti-rabbit IgG diluted 1:1000 in assay buffer). Thereafter, an Europium-labeled streptavidin (diluted 1:1000 in assay buffer) was added and incubated for 1 hour. After additional 3 washing steps, 100 μl enhancement solution was added for 30 minutes at room temperature and thereafter time-resolved fluorescence of the Europium chelate was measured using a Victor 1420 Multilabel Counter (Fa. Wallac, Turku, Finland). All measurements were done in duplicate. To measure c-fibronectin, time-resolved fluorescence immunoassay was used. Briefly, 96-well microtiter plates were coated 3 hours at room temperature with gelatin (10 μg/ml) in coating buffer (0.05 mol/L NaHCO3, pH 9.1) and thereafter blocked overnight at 4°C using assay buffer (Tris 0.05 mol/L, NaCl 0.15 mol/L, RIA grade albumin 0.5%, pH 7.7). Standards (100 μl, 5000–19 ng/ml) and culture supernatants diluted in assay buffer were added and incubated overnight at room temperature. Thereafter, the plates were incubated for 1 hour with a monoclonal mouse-anti-c-fibronectin diluted 1:1000 in assay buffer. After washing 3 times the plates were incubated for 1 hour with the second antibody (biotin-labeled anti-mouse IgG diluted 1:1000 in assay buffer) followed by 3 washing steps. Thereafter, an Europium-labeled strepavidin (diluted 1:1000 in assay buffer) was added and incubated for 1 hour. After 3 more washing steps, 100 μl enhancement solution was added for 30 minutes at room temperature and thereafter time-resolved fluorescence of the Europium chelate was measured using a Victor 1420 Multilabel Counter. All measurements were done in duplicate. TGFβ1 was measured by time-resolved fluorescence immunoassay. Ninety-six-well microtiter plates were coated overnight at room temperature with TGFβ1-sR-II/Fc Chimera (0.2 μg/ml) diluted in coating buffer (0.05 mol/L NaHCO3, pH 9.2). After 3 washing steps (wash buffer: PBS, Tween 20 0.05%, pH 7.4), plates were blocked during 2 hours at room temperature with blocking buffer (PBS, Tween 20 5%, sucrose 5%, NaN3 0.05%) and again washed 3 times. One-hundred-microliter standards (5 ng/ml − 156 pg/ml) diluted in DMEM with 0.1% RIA grade albumin and culture supernatants diluted in diluent (PBS, milk powder 0.5%, Tween 20 0.05%) were added and incubated for 2 hours at room temperature. After 3 washing steps, plates were incubated with biotin anti-human TGBβ1 diluted 1:250 in diluent (20 mmol/L Trizma-base, 150 mmol/L NaCl, 0.1. RIA grade albumin) for 2 hours, followed by 3 washing steps. Thereafter, an Europium-labeled streptavidin (diluted 1:1000 in diluent) was added and incubated for 1 hour. After additional 5 washing steps, 100 μl of enhancement solution were added for 30 minutes at room temperature and thereafter time-resolved fluorescence of the Europium chelate was measured using a Victor 1420 Multilabel Counter. All measurements were done in duplicate. DNA was quantified as previously described28Labarca C Paigen K A simple, rapid, and sensitive DNA assay procedure.Anal Biochem. 1980; 102: 344-352Crossref PubMed Scopus (4701) Google Scholar by fluorometry using bisbenzimide and calf thymus DNA as a standard. Fluorescence (Ex. 350 nm, Em. 450 nm) was measured with a Victor 1420 Multilabel Counter. Bromodeoxyuridine (BrdU) incorporation was quantified by time-resolved fluorescence of a Europium chelate.29Bachem MG Dietz R Gressner AM Quantitative measurement of cell proliferation using incorporation of 5-bromo-2-deoxyuridine, monoclonal antibody against 5-bromo-2-deoxyuridine (Mab anti-BrdUrd) and time-resolved fluorometry of europium chelat.Eur J Clin Chem Clin Biochem. 1995; 33 (abstract): A30Google Scholar Briefly, cells were labeled for 18 hours with BrdU (5 × 10−5 M). Thereafter, cell cultures were washed twice with TNT (0.1 Mol/L Tris-HCl, 0.15 Mol/L NaCl, 0.5% RIA grade albumin, pH 7.4), fixed using ethanol/acetic acid (95/5, v/v), and then incubated for 20 minutes at 4°C with 0.05 mol/L HCl. After another washing step, DNA was cleaved by incubation for 45 minutes at 80°C with formamide/trisodium citrate (88 mg trisodium citrate in 38 ml formamide). After 2 washing steps, nonspecific binding was blocked by incubation with FCS (diluted 1:1 with TNB: 0.1 mol/L Tris-HCl, 0.15 mol/L NaCl, 0.05% Tween 20, pH 7.4) followed by 3 washing steps. Thereafter, first antibody (mouse anti-BrdU IgG diluted 1:500 in TNB. was added and incubated with gentle shaking for 2 hours at 22°C. After 3 more washing steps, a second antibody (biotin-labeled anti-mouse IgG, dilu
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