Activation of Rat Alveolar Macrophage-Derived Latent Transforming Growth Factor β-1 by Plasmin Requires Interaction with Thrombospondin-1 and its Cell Surface Receptor, CD36
1999; Elsevier BV; Volume: 155; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)65183-8
ISSN1525-2191
AutoresTeshome Yehualaeshet, Robert O’Connor, Julia M. Green-Johnson, Sabine Mai, Roy L. Silverstein, Joanne E. Murphy-Ullrich, Nasreen Khalil,
Tópico(s)Blood Coagulation and Thrombosis Mechanisms
ResumoTransforming growth factor-β-1 (TGF-β1) is secreted by cells in a latent form (L-TGF-β1) noncovalently bound to a latency-associated peptide. Activated alveolar macrophages obtained from rat lungs after bleomycin-induced pulmonary injury released increased amounts of active TGF-β1 as well as plasmin, a protease, and thrombospondin-1 (TSP-1), a trimeric glycoprotein. Previously we had demonstrated that plasmin was critical to the activation of L-TGF- β1. In the present study we demonstrated that TSP-1 is also important for the activation of L-TGF- β1 because the activation can be inhibited by anti-TSP-1 monoclonal antibody. Proteins obtained from alveolar macrophage cell lysates immunoprecipitated with antibodies specific for TSP-1 were identified on immunoblots as LAP and TGF-β1, indicating that TSP-1/L-TGF-β1 complexes are present on alveolar macrophages. However, in the presence of plasmin both latency-associated peptide and TGF-β1 were decreased in the same cell lysates, indicating that L-TGF-β1 associated with TSP-1 is released by plasmin. Using immunofluorescence and antibodies to TGF-β1 and CD36, a receptor for TSP-1, there was colocalization of TGF-β1 with CD36. Because TSP-1 but not TGF-β1 is a natural ligand for CD36, these findings suggest that the L-TGF-β1 in a complex with TSP-1 localizes to the macrophage cell surface when TSP-1 interacts with its receptor, CD36. Furthermore, the association of TSP-1/L-TGF-β1 complex with CD36 is necessary to the activation of L-TGF-β1 because antibodies to CD36 prevent the colocalization of TGF-β1 with CD36 as observed by immunofluorescence and inhibit activation of the L-TGF-β1 by explanted alveolar macrophages. These findings suggest that activation of L-TGF-β1 by plasmin occurs at the cell surface of activated alveolar macrophages and requires a TSP-1/CD36 interaction. Transforming growth factor-β-1 (TGF-β1) is secreted by cells in a latent form (L-TGF-β1) noncovalently bound to a latency-associated peptide. Activated alveolar macrophages obtained from rat lungs after bleomycin-induced pulmonary injury released increased amounts of active TGF-β1 as well as plasmin, a protease, and thrombospondin-1 (TSP-1), a trimeric glycoprotein. Previously we had demonstrated that plasmin was critical to the activation of L-TGF- β1. In the present study we demonstrated that TSP-1 is also important for the activation of L-TGF- β1 because the activation can be inhibited by anti-TSP-1 monoclonal antibody. Proteins obtained from alveolar macrophage cell lysates immunoprecipitated with antibodies specific for TSP-1 were identified on immunoblots as LAP and TGF-β1, indicating that TSP-1/L-TGF-β1 complexes are present on alveolar macrophages. However, in the presence of plasmin both latency-associated peptide and TGF-β1 were decreased in the same cell lysates, indicating that L-TGF-β1 associated with TSP-1 is released by plasmin. Using immunofluorescence and antibodies to TGF-β1 and CD36, a receptor for TSP-1, there was colocalization of TGF-β1 with CD36. Because TSP-1 but not TGF-β1 is a natural ligand for CD36, these findings suggest that the L-TGF-β1 in a complex with TSP-1 localizes to the macrophage cell surface when TSP-1 interacts with its receptor, CD36. Furthermore, the association of TSP-1/L-TGF-β1 complex with CD36 is necessary to the activation of L-TGF-β1 because antibodies to CD36 prevent the colocalization of TGF-β1 with CD36 as observed by immunofluorescence and inhibit activation of the L-TGF-β1 by explanted alveolar macrophages. These findings suggest that activation of L-TGF-β1 by plasmin occurs at the cell surface of activated alveolar macrophages and requires a TSP-1/CD36 interaction. At sites of lung injury before connective tissue synthesis there is an influx of activated macrophages.1Rappolee DA Werb Z Macrophage derived growth factors.Curr Topics Microbiol Immumol. 1992; 181: 87-140PubMed Google Scholar, 2Stein M Keshav A The versatility of macrophages.Clin Exp Allergy. 1992; 22: 19-27Crossref PubMed Scopus (67) Google Scholar When activated, macrophages secrete a number of pro-inflammatory and fibrogenic cytokines.1Rappolee DA Werb Z Macrophage derived growth factors.Curr Topics Microbiol Immumol. 1992; 181: 87-140PubMed Google Scholar, 2Stein M Keshav A The versatility of macrophages.Clin Exp Allergy. 1992; 22: 19-27Crossref PubMed Scopus (67) Google Scholar Of these cytokines, transforming growth factor-β-1 (TGF-β1), a multifunctional peptide, is one of the most potent regulators of inflammation and connective tissue synthesis.3Wahl SM Transforming growth factor β: the good, the bad and the ugly.J Exp Med. 1994; 108: 1587-1590Crossref Scopus (433) Google Scholar TGF-β1 is synthesized as a large 390-amino acid precursor that undergoes a number of intracellular processing steps, including cleavage of the latent-associated peptide (LAP), to produce the mature 25-kd TGF-β1 protein.4Gleizes PE Munger JS Nunes I Harpel JG Mazzieri R Noguera S Rifkin DB TGF-β latency: biological significance and mechanism of activation.Stem Cells. 1997; 15: 190-197Crossref PubMed Scopus (222) Google Scholar However, with rare exceptions, when TGF-β1 is secreted by cells it remains noncovalently associated in a 1:1 ratio with its LAP.4Gleizes PE Munger JS Nunes I Harpel JG Mazzieri R Noguera S Rifkin DB TGF-β latency: biological significance and mechanism of activation.Stem Cells. 1997; 15: 190-197Crossref PubMed Scopus (222) Google Scholar The noncovalent association of TGF-β1 with its LAP renders the TGF-β1 unable to interact with its receptor and, therefore, biologically inactive.4Gleizes PE Munger JS Nunes I Harpel JG Mazzieri R Noguera S Rifkin DB TGF-β latency: biological significance and mechanism of activation.Stem Cells. 1997; 15: 190-197Crossref PubMed Scopus (222) Google Scholar TGF- β1 and its receptors are ubiquitously expressed, and, because TGF-β1 has numerous biological effects, the ability of a cell to activate L-TGF-β1 on secretion may be an important regulatory mechanism of TGF- β1 action in vivo. We had previously demonstrated that after pulmonary injury induced by the antineoplastic antibiotic bleomycin, explanted alveolar macrophages generated maximal quantities of biologically active TGF-β1 and plasmin 7 days after bleomycin induced pulmonary injury.5Khalil N Corne S Whitman C Yacyshyn H Plasmin regulates the activation of cell associated latent TGF-β1 secreted by rat alveolar macrophages after in vivo bleomycin injury.Am J Resp Mol Cell Biol. 1996; 15: 252-259Crossref PubMed Scopus (129) Google Scholar Furthermore, the secretion of active TGF-β1 was totally inhibited by the presence of α2-antiplasmin, a naturally occurring inhibitor of plasmin.5Khalil N Corne S Whitman C Yacyshyn H Plasmin regulates the activation of cell associated latent TGF-β1 secreted by rat alveolar macrophages after in vivo bleomycin injury.Am J Resp Mol Cell Biol. 1996; 15: 252-259Crossref PubMed Scopus (129) Google Scholar When large quantities of plasmin were added to activated alveolar macrophages, there was further activation of the L-TGF-β1.5Khalil N Corne S Whitman C Yacyshyn H Plasmin regulates the activation of cell associated latent TGF-β1 secreted by rat alveolar macrophages after in vivo bleomycin injury.Am J Resp Mol Cell Biol. 1996; 15: 252-259Crossref PubMed Scopus (129) Google Scholar However, when plasmin was added to the L-TGF-β1 in cell-free CM, no 9further activation of L-TGF-β1 occurred.5Khalil N Corne S Whitman C Yacyshyn H Plasmin regulates the activation of cell associated latent TGF-β1 secreted by rat alveolar macrophages after in vivo bleomycin injury.Am J Resp Mol Cell Biol. 1996; 15: 252-259Crossref PubMed Scopus (129) Google Scholar Our findings suggested that the generation of plasmin is important in the posttranslational activation of alveolar macrophage-derived L-TGF- β1 during an inflammatory pulmonary injury response and that the activation requires the presence of intact macrophages.5Khalil N Corne S Whitman C Yacyshyn H Plasmin regulates the activation of cell associated latent TGF-β1 secreted by rat alveolar macrophages after in vivo bleomycin injury.Am J Resp Mol Cell Biol. 1996; 15: 252-259Crossref PubMed Scopus (129) Google Scholar In this paper, we demonstrate that alveolar macrophages also secrete increased quantities of TSP-1, a glycoprotein previously reported to activate L-TGF-β1 both in the presence of cells and in cell-free solution.6Schultz-Cherry S Murphy-Ullrich JE Thrombospondin causes activation of latent transforming growth factor-β secreted by endothelial cells by a novel mechanism.J Cell Biol. 1993; 122: 923-932Crossref PubMed Scopus (401) Google Scholar When alveolar macrophages were cultured in the presence of neutralizing antibodies to TSP-1, the posttranslational activation of L-TGF-β1 was abrogated. Furthermore, antibodies to the TSP-1 receptor, CD36, also abrogated activation of alveolar macrophage-derived L-TGF-β1. Our findings support a model in which L-TGF-β1 is held at the cell surface by a TSP-1/CD36 interaction and is processed by plasmin generated by activated alveolar macrophages. Female Sprague-Dawley rats, which were free of respiratory disease and weighed between 250 and 300 g, were obtained from the University of Manitoba vivarium. In each experiment, all rats were matched for age and weight. Bleomycin (Blenoxane) was a gift from Bristol-Myers Squibb (Evansville, IN). Neutralizing antibody to TGF-β1–3 was obtained from Genzyme (Cambridge, MA). Antibody to TGF-β1 used for Western blot analysis was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The recombinant anti-human LAP antibody was obtained from R&D Systems (Minneapolis, MN). TSP-1 depleted of TGF-β activity (sTSP-1. and monoclonal antibodies to human platelet TSP-1 depleted of associated TGF- β1 (mAb 133) used in the enzyme-linked immunosorbent assay (ELISA) and TSP-1 immunoprecipitates were either used by or obtained from Dr. Murphy-Ullrich.6Schultz-Cherry S Murphy-Ullrich JE Thrombospondin causes activation of latent transforming growth factor-β secreted by endothelial cells by a novel mechanism.J Cell Biol. 1993; 122: 923-932Crossref PubMed Scopus (401) Google Scholar, 7Murphy-Ullrich JC Schultz-Cherry S Hook M Transforming growth factor-β complexes with thrombospondin.Mol Biol Cell. 1992; 3: 181-188Crossref PubMed Scopus (222) Google Scholar Anti-TSP-1 antibody used in experiments to neutralize TSP-1 from activated alveolar macrophages was obtained from Sigma (St. Louis, MO). The CD36 antibody, 5F1, was provided by the Fifth International Workshop on Leukocyte Differentiation Antigens.8Silverstein RL La Salla J Pearce SE CD36 cluster report. Leucocyte Typing V: White Cell Differentiation Antigens.in: Schlossman SF Boumsell L Gilks W Harlan JM Kishimoto T Morimoto C Ritz O Silverstein RL Springer TA Tedder TF Todd RF Oxford University Press, Oxford1995: 1274-1275Google Scholar This procedure is described in detail elsewhere.5Khalil N Corne S Whitman C Yacyshyn H Plasmin regulates the activation of cell associated latent TGF-β1 secreted by rat alveolar macrophages after in vivo bleomycin injury.Am J Resp Mol Cell Biol. 1996; 15: 252-259Crossref PubMed Scopus (129) Google Scholar, 9Khalil N Bereznay O Sporn MB Greenberg AH Macrophage production of transforming growth factor-β and fibroblast collagen synthesis in chronic pulmonary inflammation.J Exp Med. 1989; 170: 727-737Crossref PubMed Google Scholar, 10Khalil N Whitman C Zuo L Danielpour D Greenberg AH Regulation of alveolar macrophage transforming growth factor-β secretion by corticosteroids in bleomycin-induced pulmonary inflammation in the rat.J Clin Invest. 1993; 92: 1812-1818Crossref PubMed Scopus (169) Google Scholar For some experiments rats were sacrificed at several time intervals after bleomycin or normal saline treatment,5Khalil N Corne S Whitman C Yacyshyn H Plasmin regulates the activation of cell associated latent TGF-β1 secreted by rat alveolar macrophages after in vivo bleomycin injury.Am J Resp Mol Cell Biol. 1996; 15: 252-259Crossref PubMed Scopus (129) Google Scholar, 9Khalil N Bereznay O Sporn MB Greenberg AH Macrophage production of transforming growth factor-β and fibroblast collagen synthesis in chronic pulmonary inflammation.J Exp Med. 1989; 170: 727-737Crossref PubMed Google Scholar, 10Khalil N Whitman C Zuo L Danielpour D Greenberg AH Regulation of alveolar macrophage transforming growth factor-β secretion by corticosteroids in bleomycin-induced pulmonary inflammation in the rat.J Clin Invest. 1993; 92: 1812-1818Crossref PubMed Scopus (169) Google Scholar whereas for other experiments alveolar macrophages were harvested 7 days after bleomycin administration. The latter time point was used based on our findings that alveolar macrophages are maximally stimulated at this time to secrete active TGF-β1.5Khalil N Corne S Whitman C Yacyshyn H Plasmin regulates the activation of cell associated latent TGF-β1 secreted by rat alveolar macrophages after in vivo bleomycin injury.Am J Resp Mol Cell Biol. 1996; 15: 252-259Crossref PubMed Scopus (129) Google Scholar This procedure is described in detail elsewhere.5Khalil N Corne S Whitman C Yacyshyn H Plasmin regulates the activation of cell associated latent TGF-β1 secreted by rat alveolar macrophages after in vivo bleomycin injury.Am J Resp Mol Cell Biol. 1996; 15: 252-259Crossref PubMed Scopus (129) Google Scholar, 10Khalil N Whitman C Zuo L Danielpour D Greenberg AH Regulation of alveolar macrophage transforming growth factor-β secretion by corticosteroids in bleomycin-induced pulmonary inflammation in the rat.J Clin Invest. 1993; 92: 1812-1818Crossref PubMed Scopus (169) Google Scholar The lungs were lavaged to obtain cells for culture of alveolar macrophages. Alveolar macrophages were maintained in serum-free media containing Gentamicin (4 mg/100 ml; Roussel, Montreal, PQ), Fungizone (100 μl/100 ml; Gibco BRL, Grand Island, NY) and 0.2% clotted bovine calf plasma (BCP; National Biological Laboratory Limited, Dugald, MB). The macrophages were cultured in the absence or presence of a number of reagents consisting of anti-TSP-1 antibody, 5F1 (anti-CD36 antibody), CD36 synthetic peptide (aa 93–110), or sTSP-1. In experiments to determine the effects of exogenous sTSP-1, alveolar macrophages were cultured with varying quantities of sTSP-1 for 2 hours before the collection of conditioned media (CM). In addition, CM from the same alveolar macrophages cultured in parallel was incubated in a cell-free solution with sTSP-1 for 2 hours. Incubation of CM with sTSP-1 for 2 hours was chosen based on our previous findings, that sTSP-1 can activate L-TGF-β1 within 2 hours.11Schultz-Cherry S Lawler J Murphy-Ullrich JE The type-1 repeats of thrombospondin-1 activates latent transforming growth factor-β.J Biol Chem. 1994; 269: 26783-26788Abstract Full Text PDF PubMed Google Scholar In some experiments the cells were cultured in the absence or presence of 5F1, the CD36 antibody, before the addition of sTSP-1. In experiments to determine whether or not both plasmin and TSP-1 are required to activate alveolar macrophage-derived L-TGF-β1, the alveolar macrophages were cultured with sTSP-1 in the absence or presence of aprotinin, an inhibitor of plasmin activity.12Witman B On the reaction of plasmin or plasmin-streptokinase complex with aprotinin or α2-antiplasmin.Thromb Res. 1980; 17: 143-152Abstract Full Text PDF PubMed Scopus (52) Google Scholar After 20 hours of incubation in 5% CO2 at 37°C, the media was collected in the presence of protease inhibitors (leupeptin 0.5 μg/ml, Amersham, Poole, UK; aprotinin 1 μg/ml, and pepstatin 1 μg/ml, both from Sigma, Oakville, ON), and frozen at −80°C until ready for TGF-β quantitation.5Khalil N Corne S Whitman C Yacyshyn H Plasmin regulates the activation of cell associated latent TGF-β1 secreted by rat alveolar macrophages after in vivo bleomycin injury.Am J Resp Mol Cell Biol. 1996; 15: 252-259Crossref PubMed Scopus (129) Google Scholar, 10Khalil N Whitman C Zuo L Danielpour D Greenberg AH Regulation of alveolar macrophage transforming growth factor-β secretion by corticosteroids in bleomycin-induced pulmonary inflammation in the rat.J Clin Invest. 1993; 92: 1812-1818Crossref PubMed Scopus (169) Google Scholar The CCL-64 growth inhibition assay to identity and quantitate TGF-β has been described.5Khalil N Corne S Whitman C Yacyshyn H Plasmin regulates the activation of cell associated latent TGF-β1 secreted by rat alveolar macrophages after in vivo bleomycin injury.Am J Resp Mol Cell Biol. 1996; 15: 252-259Crossref PubMed Scopus (129) Google Scholar, 9Khalil N Bereznay O Sporn MB Greenberg AH Macrophage production of transforming growth factor-β and fibroblast collagen synthesis in chronic pulmonary inflammation.J Exp Med. 1989; 170: 727-737Crossref PubMed Google Scholar, 10Khalil N Whitman C Zuo L Danielpour D Greenberg AH Regulation of alveolar macrophage transforming growth factor-β secretion by corticosteroids in bleomycin-induced pulmonary inflammation in the rat.J Clin Invest. 1993; 92: 1812-1818Crossref PubMed Scopus (169) Google Scholar, 13Danielpour D Hart LL Flanders KC Roberts AB Sporn MB Immunodetection and quantitation of the two forms of transforming growth factor-β (TGF-β1 and TGF-β2) secreted by cells in culture.J Cell Physiol. 1989; 138: 78-86Crossref Scopus (418) Google Scholar. Briefly, to subconfluent cells in 0.2% BCP and resuspended in α-MEM, 0.2. BCP, 10 mmol/L Hepes at pH 7.4, penicillin (25 μg/ml) and streptomycin (25 μg/ml), and cultured as 5 × 104 cells per 0.5 ml in 24-well costar dishes (Flow Laboratories, Inc., Mississauga, ON) was added neutral CM or CM that was acidified and subsequently neutralized. After 22 hours the cells were pulsed with 0.25 Ci of 5-[125I] iodo 2′-deoxyuridine (ICN Pharmaceutical, Costa Mesa, CA) for 2 to 3 hours at 37°C and eventually lysed with 1 ml of 1 N NaOH for 30 minutes at room temperature and the 125I-UdR was counted in a γ counter (LKB Instruments, Gaithersburg, MD). A standard curve using porcine TGF-β1 was included in each assay. For confirmation of TGF-β activity, neutralizing monoclonal antibody to TGF-β1–3 (Genzyme) was added before the addition of the CM5Khalil N Corne S Whitman C Yacyshyn H Plasmin regulates the activation of cell associated latent TGF-β1 secreted by rat alveolar macrophages after in vivo bleomycin injury.Am J Resp Mol Cell Biol. 1996; 15: 252-259Crossref PubMed Scopus (129) Google Scholar, 9Khalil N Bereznay O Sporn MB Greenberg AH Macrophage production of transforming growth factor-β and fibroblast collagen synthesis in chronic pulmonary inflammation.J Exp Med. 1989; 170: 727-737Crossref PubMed Google Scholar, 10Khalil N Whitman C Zuo L Danielpour D Greenberg AH Regulation of alveolar macrophage transforming growth factor-β secretion by corticosteroids in bleomycin-induced pulmonary inflammation in the rat.J Clin Invest. 1993; 92: 1812-1818Crossref PubMed Scopus (169) Google Scholar, 13Danielpour D Hart LL Flanders KC Roberts AB Sporn MB Immunodetection and quantitation of the two forms of transforming growth factor-β (TGF-β1 and TGF-β2) secreted by cells in culture.J Cell Physiol. 1989; 138: 78-86Crossref Scopus (418) Google Scholar and resulted in abrogation of all TGF-β activity. The wells of 96-well plates (Falcon tissue culture plates) were coated with 200 ng/well of the mAb 133 (anti-sTSP-1). For each experiment, a standard curve containing wells with 200 μl of CM and several concentrations of sTSP-1, ranging from 15–150 ng/well, was included. To quantitate TSP-1 in CM, 200 μl of alveolar macrophage derived CM in carbonate buffer, pH 9.6, was incubated overnight at 4°C. The next day, the wells were washed three times with PBS, 0.05%, Tween −20. Nonspecific binding sites were blocked by incubating with 250 μl/well of 1% BSA for 1 hour at 37°C. The wells were then washed with PBS and incubated with 200 μl/well of mAb 133 (7.5 μg/ml) in PBS-Tween for 90 minutes at 37°C. The wells were then washed and incubated with 30 ng/ml of alkaline phosphatase-labeled goat anti-mouse lgG for 90 minutes at 37°C, and then assayed for color development using the Sigma 104 AP substrate. Color development was stopped by adding 50 μl of 2N NaOH, and absorbency at 405 nm was read using a Bio-Tek ELISA reader. The CD36 peptide, YRVRFLAKENVTQDAEDNC(93–110) was synthesized, based on the work of Leung et al.14Leung LL Wei-Xing L McGregor JL Albrecht G Howard RJ CD36 peptides enhance or inhibit CD36 thrombospondin binding: a two-step process of ligand receptor interaction.J Biol Chem. 1992; 267: 18244-18250Abstract Full Text PDF PubMed Google Scholar The peptide was synthesized with an Applied Biosystems model 431A peptide synthesizer using Fmoc (N-(9-Fluoreny D-methoxycarbonyl) chemistry and purified by reverse high pressure liquid chromatography, using a C18 column. Alveolar macrophages were obtained by bronchoalveolar lavage 7 days after intratracheal normal saline or bleomycin administration, and were adjusted to 1 × 106 cells/ml. For some experiments, alveolar macrophages obtained after bleomycin administration were cultured in the presence of anti-CD36 antibody (20 μg per 106 macrophages) for 30 minutes before the immunofluoresence procedure. The immunofluorescence analysis was performed as previously described.15Mai S Overexpression of C-myc precedes amplification of gene encoding dihydrofolate reductase.Gene. 1994; 148: 253-260Crossref PubMed Scopus (45) Google Scholar Briefly, cytospin smears of 1 × 105 cells suspension were fixed with 3.7% formaldehyde for 10 minutes and washed with PBS. Nonspecific binding was blocked by 100% lamb serum (Gibco BRL) for 5 minutes. The cells were then incubated with anti-TGF-β1 (Santa Cruz Biotechnology) and anti-CD36 antibody, both at a concentration of 1 μg/ml, for 45 minutes. After washing with PBS the cells were incubated for 30 minutes with polyclonal anti-rabbit antibody conjugated to tetramethylrhodamine-isothiocyanate (TRITC) or conjugated with fluorescein isothiocyanate (FITC) monoclonal anti-mouse IgM as secondary antibodies for the detection of TGF-β1 and CD36 antibodies, respectively. Both secondary antibodies were used as 1 μg/ml. The slides were washed three times with PBS. Nuclear staining was done using 4′, 6′ Diamidino-2-phenylindole (DAPI) at a concentration of 1 μg/ml for 5 minutes. The slides were mounted in anti-bleach (12. glycerol, 4.8% Mowiol 4–88 (Hoechst), 2.4% DABCO (1, 4 Diazabicylo [2.2.2]-octane (Fluka) in 0.2 Mol/L Tris/HCl, pH = 8.5). Image analysis was performed using a Zeiss Axiophot microscope, equipped with a cooled CCD camera CH 250/a (Optikon/Photometrics), driven by IPLabs Spectrum software version 3.1, Signal Analytics). For TRITC-conjugated antibodies, the filter combination was BP540/FT580/LP590, resulting in a red emission identifying the location of TGF-β1on the macrophage surface. For the FITC-conjugated antibody, the filter combination was 450–490/FT510/515–565, resulting in a green emission identifying the location of CD36 on the macrophage surface. For DAPI, the filter combination was G365/FT395/LP420, resulting in a blue emission. To determine whether TGF-β1 and CD36 were colocalized, the images were obtained by a pixel overlap, which was achieved with IPLab Spectrum/Multiprobe (V.3.1, Signal Analytics, Fairfax, VA). In areas where the TGF-β1 was localized to the same region as CD36, the emission was yellow. Alveolar macrophages obtained from rats that had been treated with normal saline or bleomycin 7 days earlier were lysed by incubating the cells for 20 minutes on ice in a RIPA lysis buffer (50 mmol/L Tris-Cl, pH 7.5; 150 mmol/L NaCl, 1% Nonidet P-40, 0.1% sodium deoxycholate and a cocktail of protease inhibitors; phenylmethylsulfonyl fluoride 1 mmol/L, leupeptin 1 μg/ml, and aprotinin 0.1 μg/ml, all from Sigma). In some experiments the alveolar macrophages were cultured overnight in the absence or presence of α2-antiplasmin or aprotinin (Sigma), both inhibitors of plasmin activity.12Witman B On the reaction of plasmin or plasmin-streptokinase complex with aprotinin or α2-antiplasmin.Thromb Res. 1980; 17: 143-152Abstract Full Text PDF PubMed Scopus (52) Google Scholar The lysate was centrifuged for 20 minutes at 12,000 × g at 4°C, the supernatant collected, and the total protein content determined by the Bradford dye-binding assay (Bio-Rad, Mississauga, ON). The total protein extract (300 μg) and 10 μg of anti-sTSP-1 antibody (mAb 133) or IgG as an isotype control for the anti-sTSP-1 antibody was incubated overnight at 4°C. After a further incubation of 2 hours with 30 μl of Protein G Plus/Protein A-Agarose (Calbiochem, San Diego, CA) the immune complexed beads were collected. The beads were washed four times with RIPA buffer and placed in a final suspension with 25 μl of Laemmli buffer and boiled for 10 minutes. The supernatant containing the precipitated proteins was then used for Western blot analysis. The protein samples (25 μl) were electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in a MiniPROTEAN II Electrophoresis Cell (Bio-Rad, Hercules, CA). Rainbow colored protein molecular weight markers (Amersham) were run parallel to each blot as an indicator of the molecular weight. Equality in loading of protein was evaluated using silver staining (not shown). The separated proteins were transferred at 50 V overnight onto nitrocellulose membrane (Gibco BRL) in a Mini trans-Blot chamber with transfer buffer (25 mmol/L Tris Cl, 192 mmol/L glycine, and 20% methanol). The nitrocellulose membrane was blocked for 1 hour by using 5% instant skim milk powder in TBS. For detection of LAP a 1:500 dilution of anti-recombinant human LAP antibody (anti-rh LAP; R&D Systems) in 1% instant skim milk powder was used. After washing, the nitrocellulose membrane was incubated with horseradish peroxidase linked with the secondary antibody (Goat anti-mouse IgG; Bio-Rad) as recommended by the manufacturer. Finally the washed blots were exposed to enhanced chemiluminescence detection system (Amersham) and recorded on an autoradiograph (Kodak X-Omat film). Before reprobing, the nitrocellulose membrane was incubated at 50°C for 30 minutes with a stripping buffer (100 mmol/L 2-mercaptoethanol, 2% sodium dodecyl sulfate, and 62.5 mmol/L Tris-HCl, pH 6.7). The blots were rinsed twice with TBS. To ensure the removal of antibodies, membranes were incubated with the enhanced chemiluminescence detection reagents and exposed to film (Kodak). No band was detected, confirming that all antibodies were stripped off the membrane. The same nitrocellulose membrane was blocked using 5% instant skim milk powder in TBS. For detection of TGF-β1, 0.5 μg/ml of rabbit polyclonal IgG TGF-β1 antibody (Santa Cruz Biotechnology) was used as above. Statistical analysis using analysis of variance was done by Dr. Bob Tate, Biostatistical Unit, University of Manitoba. In addition to plasmin, TSP-1, a large trimeric glycoprotein,16Bornstein P Thrombospondins: structure and regulation of expression.FASEB J. 1992; 6: 3290-3299Crossref PubMed Scopus (306) Google Scholar had also been demonstrated to be a physiological substance that can activate L-TGF-β1.6Schultz-Cherry S Murphy-Ullrich JE Thrombospondin causes activation of latent transforming growth factor-β secreted by endothelial cells by a novel mechanism.J Cell Biol. 1993; 122: 923-932Crossref PubMed Scopus (401) Google Scholar, 17Yee JA Yan L Dominguez JC Allan EH Martin TJ Plasminogen-dependent activation of latent transforming growth factor β (TGF-β) by growing cultures of osteoblast-like cells.J Cell Physiol. 1993; 157: 528-534Crossref PubMed Scopus (108) Google Scholar TSP-1 was constitutively secreted by alveolar macrophages, but after bleomycin injury, TSP-1 secretion was further increased and reached maximal levels 7 days after bleomycin administration (Figure 1A). The secretion of TSP-1 declined rapidly thereafter, and by 28 days after bleomycin administration, the secretion returned to that of control levels of alveolar macrophages from normal saline-treated rats. To determine whether the presence of TSP-1 in the CM was necessary for the activation of L-TGF-β1, alveolar macrophages were cultured in the absence or presence of anti-TSP-1 monoclonal antibody. Anti-TSP-1 antibody inhibited the activation of L-TGF- β1 but had no effect on the secretion of the latent form of TGF-β1 (Figure 1B) by the alveolar macrophages. Neutral CM of alveolar macrophages activated by in vivo bleomycin injury contained 67.6 ± 7.9 pg of TGF-β1 per 106 cells. After the addition of 50 μg/ml of the isotype control for anti-TSP-1, 50.13 ± 13.4 pg of TGF-β1 per 106 was present in neutral CM (P < 0.934). Because TSP-1 has been reported to activate L-TGF-β1 in solution,6Schultz-Cherry S Murphy-Ullrich JE Thrombospondin causes activation of latent transforming growth factor-β secreted by endothelial cells by a novel mechanism.J Cell Biol. 1993; 122: 923-932Crossref PubMed Scopus (401) Google Scholar we next determined if alveolar macrophage-derived L-TGF-β1 could be directly activated in CM by sTSP-1. After 20 hours in culture, activated alveolar macrophages secreted large quantities of L-TGF-β1.5Khalil N Corne S Whitman C Yacyshyn H Plasmin regulates the activation of cell associated latent TGF-β1 secreted by rat alveolar macrophages after in vivo bleomycin injury.Am J Resp Mol Cell Biol. 1996; 15: 252-259Crossref PubMed Scopus (129) Google Scholar The addition of sTSP-1 to this cell-free L-TGF-β1 unexpectedly diminished the quantity of active TGF-β1 that could be detected by our bioassay (Figure 1C). In contrast, sTSP-1 added directly to the same CM but, in the presence of alveolar macrophages, further activated L-TGF-β1 (Figure 1C). These findings demonstrate that TSP-1 is effective in promoting the activation
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