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• W2080798474 abstract "Fibrosis results from inflammatory tissue damage and impaired regeneration. In the context of bleomycin-induced pulmonary fibrosis, we demonstrated that the matricellular protein termed secreted protein acidic and rich in cysteine (SPARC) distinctly regulates inflammation and collagen deposition, depending on its cellular origin. Reciprocal Sparc−/− and wild-type (WT) bone marrow chimeras revealed that SPARC expression in host fibroblasts is required and sufficient to induce collagen fibrosis in a proper inflammatory environment. Accordingly, Sparc−/− >WT chimeras showed exacerbated inflammation and fibrosis due to the inability of Sparc−/− macrophages to down-regulate tumor necrosis factor production because of impaired responses to tumor growth factor-β. Hence, the use of bone marrow cells expressing a dominant-negative form of tumor growth factor-β receptor type II under the monocyte-specific CD68 promoter, as a decoy, phenocopied Sparc−/− donor chimeras. Our results point to an unexpected dual role of SPARC in oppositely influencing the outcome of fibrosis. Fibrosis results from inflammatory tissue damage and impaired regeneration. In the context of bleomycin-induced pulmonary fibrosis, we demonstrated that the matricellular protein termed secreted protein acidic and rich in cysteine (SPARC) distinctly regulates inflammation and collagen deposition, depending on its cellular origin. Reciprocal Sparc−/− and wild-type (WT) bone marrow chimeras revealed that SPARC expression in host fibroblasts is required and sufficient to induce collagen fibrosis in a proper inflammatory environment. Accordingly, Sparc−/− >WT chimeras showed exacerbated inflammation and fibrosis due to the inability of Sparc−/− macrophages to down-regulate tumor necrosis factor production because of impaired responses to tumor growth factor-β. Hence, the use of bone marrow cells expressing a dominant-negative form of tumor growth factor-β receptor type II under the monocyte-specific CD68 promoter, as a decoy, phenocopied Sparc−/− donor chimeras. Our results point to an unexpected dual role of SPARC in oppositely influencing the outcome of fibrosis. Tissue remodeling and repair are characterized by epithelial to mesenchymal transition. Excessive inflammatory responses in tissues may lead to prominent cell destruction that exceeds the intrinsic regenerative potential of the parenchyma stem cell reservoir. Subsequently, fibrotic tissue can occupy the area of impaired regeneration. Although this form of repair does not replace the tissue or organ function (eg, myocardial scarring after infarction), the process is, in most cases, controlled and its regulation dependent on the resolution of the underlying inflammatory spur. The matricellular glycoprotein termed secreted protein acidic and rich in cysteine (SPARC) has a key role in extracellular matrix (ECM) assembly and molding. Consistently, SPARC has been implicated in the pathogenesis of several fibrotic disorders, such as adipose tissue fibrosis,1Kos K. Wilding J.P. SPARC: a key player in the pathologies associated with obesity and diabetes.Nat Rev Endocrinol. 2010; 6: 225-235Crossref PubMed Scopus (114) Google Scholar hepatic fibrosis,2Nakatani K. Seki S. Kawada N. Kitada T. Yamada T. Sakaguchi H. Kadoya H. Ikeda K. Kaneda K. Expression of SPARC by activated hepatic stellate cells and its correlation with the stages of fibrogenesis in human chronic hepatitis.Virchows Arch. 2002; 441: 466-474Crossref PubMed Scopus (51) Google Scholar, 3Frizell E. Liu S.L. Abraham A. Ozaki I. Eghbali M. Sage E.H. Zern M.A. Expression of SPARC in normal and fibrotic livers.Hepatology. 1995; 21: 847-854PubMed Google Scholar lung fibrosis,4Demopoulos K. Arvanitis D.A. Vassilakis D.A. Siafakas N.M. Spandidos D.A. MYCL1. FHIT, SPARC, p16(INK4) and TP53 genes associated to lung cancer in idiopathic pulmonary fibrosis.J Cell Mol Med. 2002; 6: 215-222Crossref PubMed Scopus (60) Google Scholar and scleroderma.5Zhou X. Tan F.K. Guo X. Arnett F.C. Attenuation of collagen production with small interfering RNA of SPARC in cultured fibroblasts from the skin of patients with scleroderma.Arthritis Rheum. 2006; 54: 2626-2631Crossref PubMed Scopus (45) Google Scholar This notion comes from data generated in Sparc−/− mice or gene-silencing strategies in models of skin, liver, and pulmonary fibrosis. Accordingly, Sparc−/− mice were protected from bleomycin-induced pulmonary fibrosis6Strandjord T.P. Madtes D.K. Weiss D.J. Sage E.H. Collagen accumulation is decreased in SPARC-null mice with bleomycin-induced pulmonary fibrosis.Am J Physiol. 1999; 277: L628-L635PubMed Google Scholar and adenoviral-mediated inhibition of SPARC, or intratracheal instillation of small-interfering RNA attenuated pulmonary fibrosis in rats and mice, respectively.7Camino A.M. Atorrasagasti C. Maccio D. Prada F. Salvatierra E. Rizzo M. Alaniz L. Aquino J.B. Podhajcer O.L. Silva M. Mazzolini G. Adenovirus-mediated inhibition of SPARC attenuates liver fibrosis in rats.J Gene Med. 2008; 10: 993-1004Crossref PubMed Scopus (53) Google Scholar, 8Wang J.C. Lai S. Guo X. Zhang X. de Crombrugghe B. Sonnylal S. Arnett F.C. Zhou X. Attenuation of fibrosis in vitro and in vivo with SPARC siRNA.Arthritis Res Ther. 2010; 12: R60Crossref PubMed Scopus (67) Google Scholar In this setting, SPARC function is mainly linked to the regulation of tumor growth factor-β1 (TGF-β1) signaling and collagen production by fibroblasts. Nevertheless, contrasting results show increased lung fibrosis in Sparc−/− mice after bleomycin administration,9Savani R.C. Zhou Z. Arguiri E. Wang S. Vu D. Howe C.C. DeLisser H.M. Bleomycin-induced pulmonary injury in mice deficient in SPARC.Am J Physiol Lung Cell Mol Physiol. 2000; 279: L743-L750PubMed Google Scholar a condition that was associated with an increased inflammatory cell infiltration. We have previously shown that the absence of SPARC exacerbated contact hypersensitivity10Sangaletti S. Gioiosa L. Guiducci C. Rotta G. Rescigno M. Stoppacciaro A. Chiodoni C. Colombo M.P. Accelerated dendritic-cell migration and T-cell priming in SPARC-deficient mice.J Cell Sci. 2005; 118: 3685-3694Crossref PubMed Scopus (52) Google Scholar and favored leukocyte infiltration into the tumor parenchyma.11Sangaletti S. Stoppacciaro A. Guiducci C. Torrisi M.R. Colombo M.P. Leukocyte, rather than tumor-produced SPARC, determines stroma and collagen type IV deposition in mammary carcinoma.J Exp Med. 2003; 198: 1475-1485Crossref PubMed Scopus (120) Google Scholar Said and colleagues reported that SPARC is able to ameliorate tumor-associated inflammation in the setting of ovarian cancer.12Said N.A. Elmarakby A.A. Imig J.D. Fulton D.J. Motamed K. SPARC ameliorates ovarian cancer-associated inflammation.Neoplasia. 2008; 10: 1092-1104Abstract Full Text PDF PubMed Scopus (54) Google Scholar These findings suggest that SPARC may play contrasting roles, depending on its cellular source and the pathological setting, by either promoting or attenuating the inflammatory damage and causing parenchymal fibrosis. The aim of this work was to dissect the effector and regulatory role of SPARC in fibrotic diseases. To this end, we used the well-characterized bleomycin-induced pulmonary fibrosis model in mice, which recapitulates the disease observed in the clinical setting of patients receiving bleomycin as a chemotherapeutic agent.13Sleijfer S. Bleomycin-induced pneumonitis.Chest. 2001; 120: 617-624Crossref PubMed Scopus (475) Google Scholar This prototypical mouse model is used for studying inflammation-engendered organ fibrosis and has already generated contradictory results when Sparc−/− mice were used.6Strandjord T.P. Madtes D.K. Weiss D.J. Sage E.H. Collagen accumulation is decreased in SPARC-null mice with bleomycin-induced pulmonary fibrosis.Am J Physiol. 1999; 277: L628-L635PubMed Google Scholar, 9Savani R.C. Zhou Z. Arguiri E. Wang S. Vu D. Howe C.C. DeLisser H.M. Bleomycin-induced pulmonary injury in mice deficient in SPARC.Am J Physiol Lung Cell Mol Physiol. 2000; 279: L743-L750PubMed Google Scholar We show that SPARC, in the context of pulmonary fibrosis, exerts different functions, depending on the cell of origin. Particularly, through bone marrow transplantation (BMT) experiments, we demonstrated that SPARC produced by bone marrow–derived leukocytes limits fibrosis by reducing the extent of inflammation, whereas SPARC from fibroblasts or fibrocytes sustains fibrosis, promoting collagen assembly. We also show that the anti-inflammatory activity of SPARC is dependent on its regulation of TGF-β signaling on macrophages. To our knowledge, this is the first demonstration of the dual function of the matricellular protein SPARC in the regulation of tissue stroma homeostasis. Our observation adds a new insight into the combined and opposite anti-inflammatory and profibrotic effect of TGF-β. BALB/cAnNCrl mice, 8 to 10 weeks old, were purchased from Charles River Laboratories (Calco, Italy). CNCr.129S(B6)-Sparctm1Hwe mice were developed in our institute by backcrossing B6;129S-Sparctm1Hwe (provided by Dr. Chin Howe, Wistar Institute, Philadelphia, PA) to BALB/cAnNCrl mice for 12 generations before intercrossing them.11Sangaletti S. Stoppacciaro A. Guiducci C. Torrisi M.R. Colombo M.P. Leukocyte, rather than tumor-produced SPARC, determines stroma and collagen type IV deposition in mammary carcinoma.J Exp Med. 2003; 198: 1475-1485Crossref PubMed Scopus (120) Google Scholar Thy1a mice, on unspecified BALB/c background (originally provided by Dr. Hyam Levitsky, Johns Hopkins University, Baltimore, MD) were crossed to BALB/cAnNCrl mice at least for 6 generations. Animal experiments were authorized by the Institutional Ethical Committee for Animal Use. Mice were housed in filtered top caging, and room sentinels were checked for pathogens every 6 months by Charles River Laboratories staff. Viral pathogens that are excluded are as follows: mouse hepatitis virus), mouse parvovirus, mouse encephalomyelitis virus, pneumonia virus of mice, sendai virus, reovirus-3, Hantaan virus, lymphocytic choriomeningitis virus), mouse adenovirus, minute virus of mice, rotavirus, polyomavirus, K virus, ectromelia virus, mouse thymic virus, and mouse cytomegalovirus. Excluded bacterial agents are as follows: Mycoplasma pulmonis, cilia-associate respiratory bacillus, Citrobacter rodentium, and Salmonella sp. In addition, Helicobacter spp. and mouse norovirus are also tested in sentinels but are not excluded agents. Chimeric Sparc−/− (Thy 1b)>wild type (WT) (Thy 1a) and WT (Thy 1a)>Sparc−/− (Thy 1b) mice (hereafter referred to as WT>KO and KO>WT) were obtained as previously described.11Sangaletti S. Stoppacciaro A. Guiducci C. Torrisi M.R. Colombo M.P. Leukocyte, rather than tumor-produced SPARC, determines stroma and collagen type IV deposition in mammary carcinoma.J Exp Med. 2003; 198: 1475-1485Crossref PubMed Scopus (120) Google Scholar Engraftment was verified 6 to 8 weeks after BMT by staining peripheral blood mononuclear cells with fluorescein isothiocyanate–conjugated anti-mouse Thy 1a (CD90.1; Becton Dickinson, Franklin Lakes, NJ) and phycoerythrin-conjugated anti-mouse Thy1b (CD90.2, Becton Dickinson), as well as isotype control fluorescein isothiocyanate– and phycoerythrin-conjugated mouse IgG2a. Bone marrow cells expressing the dominant negative form of TGF-β receptor II (TGF-βRII) under the monocyte-specific CD68 promoter have been obtained by infection with the lentiviral CD68DNTGFBRII vector (see below). Dermal fibroblasts from newborn mouse skin were prepared according to the method of Regnier et al.14Regnier M. Delescluse C. Prunieras M. Studies on guinea pig skin cell cultures I: separate cultures of keratinocytes and dermal fibroblasts.Acta Derm Venereol. 1973; 53: 241-247PubMed Google Scholar The dorsal skin from 1- to 3-day-old mice was incubated in 0.2% trypsin in PBS at 37°C for 1 hour. Dermal fibroblasts were produced by digestion of muscle and dermis in HBSS solution containing 0.1% collagenase and 0.1% hyaluronidase, followed by filtration, centrifugation, and resuspension. Viable cells were plated at 2 to 5 × 106 cells/mL in 50-mm-diameter plastic Petri dishes containing Dulbecco's modified Eagle's medium were supplemented with 10% fetal calf serum. Fibroblasts from both WT and Sparc−/− mice were nontumorigenic when injected into BALB/c mice. To assess fibroblast proliferation in vitro, we used spectrophotometric assay,15Oliver M.H. Harrison N.K. Bishop J.E. Cole P.J. Laurent G.J. A rapid and convenient assay for counting cells cultured in microwell plates: application for assessment of growth factors.J Cell Sci. 1989; 92: 513-518PubMed Google Scholar using 1% methylene blue dye in 0.01M borate buffer, pH 8.5, according to Colombo et al.16Colombo M.P. Lombardi L. Stoppacciaro A. Melani C. Parenza M. Bottazzi B. Parmiani G. Granulocyte colony-stimulating factor (G-CSF) gene transduction in murine adenocarcinoma drives neutrophil-mediated tumor inhibition in vivo: neutrophils discriminate between G-CSF-producing and G-CSF-nonproducing tumor cells/.J Immunol. 1992; 149: 113-119PubMed Google Scholar Fibroblast migration toward bovine fibronectin (Sigma-Aldrich, St. Louis, MO) was evaluated by using Transwell supports (Corning, Lowell, MA) as previously described.17Sangaletti S. Di Carlo E. Gariboldi S. Miotti S. Cappetti B. Parenza M. Rumio C. Brekken R.A. Chiodoni C. Colombo M.P. Macrophage-derived SPARC bridges tumor cell-extracellular matrix interactions toward metastasis.Cancer Res. 2008; 68: 9050-9059Crossref PubMed Scopus (145) Google Scholar WT and Sparc−/− macrophages were obtained from bone marrow precursors by plating them in the presence of 10 ng/mL of granulocyte-macrophage colony-stimulating factor and 10 ng/mL of IL-4. On days 2, 4, and 6, floating cells consisting mainly of dendritic cells were eliminated and supplemented medium was replaced. On day 7, up to 90% of the adherent population consisted of macrophages as determined by flow cytometry analysis using the F4/80-specific monoclonal antibody (Caltag-Medsystems, Buckingham, UK). For the generation of CD68DNTGFBRII vector, DNA was obtained from transgenic mice expressing DNTGFBRII under the human CD2 promoter (gift of Ronald E. Gress, National Institutes of Health, Bethesda, MD). By PCR, the DNTGFBRII cassette was amplified and restriction sites for AgeI and SalI were added at 5′ and 3′ ends (forward primer: 5′-TTACCGGTAGAAGTCCCAACCCAGCTTT-3′; reverse primer: 5′-AATTGTCGACATGGCTGAGTTCGAAGA-3′). The PCR product was inserted into pRRLsinCD68GFP-hPGK-ΔNGFR-WPRE lentiviral vector by AgeI/SalI cloning substituting the GFP with the DNTGFBRII sequence. For bleomycin treatment, animals were anesthetized with i.p. injection of ketamine (100 mg/kg) and xylazine (5 mg/kg). The trachea was exposed in sterile conditions, and 0.15 U per mouse of 20 g of bleomycin or saline solution were instilled in 50 μL of final volume. The skin incision was closed and mice allowed to recover under a warming lamp. Mice were sacrificed 16 days after bleomycin instillation and their lungs removed for histologic analysis, fluorescence activated cell sorter (FACS) analysis, or measurement of the hydroxyproline (HP) content. For in vivo antibody treatment, mice were administered 200 μg of rat anti-mouse tumor necrosis factor (TNF) V1q (provided by Daniela Männel, University of Regensburg, Regensburg, Germany) every 3 days, starting 1 day before bleomycin instillation. Experiments requiring V1q administration in KO>WT chimeric mice have been performed on a total of 20 mice in two consecutive experiments. In each experiment, six mice were treated with the antibody and four mice with saline as control. Lung HP content was determined spectrophotometrically according to Kivirriko et al.18Kivirikko K.I. Laitinen O. Prockop D.J. Modifications of a specific assay for hydroxyproline in urine.Anal Biochem. 1967; 19: 249-255Crossref PubMed Scopus (971) Google Scholar Briefly, lungs were homogenized in a solution of trichloroacetic acid and centrifuged for 10 minutes at 4000 × g. The pellet was washed and hydrolyzed for 16 hours in HCl (6N) at 100°C. The HP content was assessed colorimetrically at 561 nm with p-dimethylaminobenzaldehyde, quantified in micrograms and normalized for lung weight. Data are shown as total HP content. To allow for morphologic analysis, lungs were fixed in situ by intratracheal injection of 10% neutral buffered formalin after mice sacrifice. The trachea was cannulated and the lungs fixed in situ with 10% formalin at a constant rate. The optimal instilled volume for mouse lungs was 0.3 mL for 20 g of weight.19Baker J.R. Rosenkrantz H. Volumetric instillation of fixatives and inert substances into mouse lungs.Stain Technol. 1976; 51: 107-113PubMed Google Scholar Lungs were removed, maintained 24 hours in formalin, and then embedded in paraffin. Sections 3 to4 μm thick were cut from paraffin blocks and stained with H&E, Masson's trichrome, and periodic acid-Schiff stain. Grading of the tissue damage was performed by a semiquantitative scoring system based on the following variables: thickening of the alveolar wall, extent of the interstitial inflammatory infiltrate, extent of fibroblast proliferation, extent of epithelial proliferation, extracellular collagen deposition, intracellular collagen amount, and overall extent of the parenchymal damage. Each variable was scored from 0 (normal samples) to 3, according to the severity of changes. The overall histologic damage score was calculated by summing the mean scores of each variable. All of the samples were analyzed by two of the authors with specific training in mouse pathology (S.S. and C.T.) in a blinded fashion under a Leica DM3000 optical microscope (Leica Microsystems GmbH, Wetzlar, Germany), and microphotographs were collected using a Leica DFC320 digital camera (Leica Camera AG, Solms, Germany). For histopathologic and immunofluorescence (IF) analysis, a total of 35 bleomycin-treated WT and Sparc−/− mice (divided into five experiments) were used. A total of 15 saline-treated mice per group were also analyzed (three mice per experiment). Each experiment was divided as follows: lungs from four bleomycin-treated mice were fixed in formalin and those from three mice embedded in optimal cutting temperature (OCT) compound (Sakura, Torrance, CA) and lungs from two saline-treated mice were fixed in formalin and those from one mouse in OCT as control. Histopathologic analysis and IF analysis on bone marrow chimeras were similarly performed on 30 bleomycin-treated and 15 saline-treated mice per group. Histopathologic analysis on bleomycin-treated WT mice transplanted with bone marrow cells expressing the dominant negative form of TGF-βRII under the monocyte-specific CD68 promoter has been performed on a total of 10 mice, from two independent experiments. The following antibodies have been used for IF and/or immunohistochemistry (IHC): polyclonal goat anti-SPARC antibody (AF942; R&D, Minneapolis, MN), monoclonal antibody to CD90.1 and CD90.2 (Becton Dickinson), monoclonal antibody to CD68 (Hycult Biotech, Plymouth Meeting, PA), monoclonal antibody to prosurfactant protein C (Millipore, Billerica, MA), polyclonal rabbit to anti-fibroblast specific protein 1 (FSP1) (Abcam, Cambridge, MA), anti–collagen types I and IV (Millipore), polyclonal antibody to α-smooth muscle actin (α-SMA; Sigma-Aldrich), and monoclonal rat anti-mouse panreticular fibroblast marker (clone ER-TR7; Cedarlane, Burlington, NC). WT and Sparc−/− fibroblasts were washed twice with cold PBS buffer and lysed in the RIPA buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) added with a protease inhibitor cocktail (Roche, Milan, Italy). Cell lysates were microcentrifuged at 14,000 × g for 20 minutes at 4°C and supernatants collected and stored at −80°C. Protein concentration in each sample was determined by the BCATM Protein Assay kit (Thermo Fisher Scientific, Waltham, MA). Cell lysates containing equal amounts of protein (25 μg) were analyzed by SDS-PAGE on precasted minigels (Invitrogen, Carlsbad, CA) and proteins transferred to a nitrocellulose membrane (GE Healthcare, Waukesha, WI). Nonspecific binding sites were blocked in 5% nonfat dry milk in Tris-buffered saline–Tween (100 mmol/L Tris, 0.9% NaCl, pH 7.5, 0.1% Tween 20) solution. Membrane was incubated overnight at 4°C with a polyclonal rabbit anti–collagen type I antibody (1:600; Millipore) or a polyclonal monoclonal antibody to SPARC (R&D), washed three times each for 5 minutes with Tris-buffered saline containing 0.1% Tween 20, and then incubated with horseradish peroxidase–conjugated antibody (1:2500; Zymed, San Francisco, CA) for 1 hour at room temperature. After washing, blots were developed with an enhanced chemiluminescence system (ECL-plus; GE Healthcare). For Western blot analysis of TNF, 20-μm cryostat sections were obtained from snap frozen whole-lung tissue and collected in 1.5-mL tubes. Sections were then homogenized in ice-cold lysis buffer (RIPA buffer without urea). Equal amounts of total protein (25 μg) were loaded in each lane. Blots were blocked and incubated overnight at 4°C with a monoclonal rat anti-mouse TNF antibody (Becton Dickinson). Protein bands were quantified by Image Quant 5.2 (GE Healthcare, Milan, Italy). Frozen cryostat sections were fixed in acetone, dried, and incubated 20 minutes with a blocking 10% fetal calf serum solution. Sections where then incubated 1 hour with the primary antibody, washed, and incubated 40 minutes with Alexa Fluor–conjugated secondary antibody. For IF analysis of intracellular antigens, a permeabilization step in 0.1% Tween and 1% fetal calf serum solution was performed. Slides were analyzed with a confocal microscopy (Microradiance 2000; Bio-Rad Laboratories, Hercules, CA) equipped with Ar (488 nm) and HeNe (543 nm) lasers. Confocal images (512 × 512 pixels) were obtained using a ×60 oil immersion lens and analyzed using ImagePro 6.3 software. Reported images represent extended depth of field from 15 to 16 frames in stack (0.5-μm step); a focus region was selected for maximum intensity. The pinhole diameter was regulated according to the value suggested by the acquisition software to obtain the maximum resolution power. For FACS analysis cell suspensions were obtained by lung digestion with a collagenase IV/elastase (Worthington Biochemical, Lakewood, NJ) solution for 90 minutes at 4°C. The suspensions were filtered by a cell strainer (BD, Franklin Lakes, NJ), washed, resuspended in 1× PBS, and stained with the indicated monoclonal antibodies. FACS analysis was performed using BD FACSCalibur or FACSCanto from Becton Dickinson. WT and Sparc−/− mice were treated with bleomycin or saline by intratracheal instillation and sacrificed 16 days later. Histologic analysis and collagen accumulation, evaluated as HP content, showed different outcomes in the two strains. WT mice developed foci of pulmonary fibrosis with an increased number of fibroblasts, interstitial collagen, and alveolar distortion (Figure 1A). In contrast, Sparc−/− mice developed a parenchymal damage characterized by inflammatory infiltration (Figure 1A) that was not associated with foci of fibroblast accumulation or collagen deposition. According to such pictures in Sparc−/− mice, FACS analysis revealed that nearly the 50% of the cells from bleomycin-treated lungs were CD3+ T cells (44.82% ± 5.4%), whereas only 16.53% ± 9.4% were fibroblasts (α-SMA+ or FSP1+) (see Supplemental Figure S1A at http://ajp.amjpathol.org). On the contrary, the percentage of fibroblast and CD3+ cells in the lungs from WT mice treated with bleomycin was 41.86% ± 6.31% and 21.83% ± 2.8%, respectively. Accordingly, IHC confirmed the presence of α-SMA+ cells in the fibrotic areas of WT mice, whereas in Sparc−/− mice, antibody to α-SMA stained only cells associated with bronchi or blood vessels (Figure 1A). Consistently, HP content was higher in WT than Sparc−/− mice (Figure 1B). These results suggest that in the absence of SPARC fibrosis was reduced, whereas inflammation was apparently increased. Nevertheless, SPARC expression, evaluated by confocal microscopy in bleomycin-treated lungs, was similar in an area of inflammation and fibrosis. Indeed, a SPARC-specific monoclonal antibody stained CD45+ infiltrating leukocytes, CD68+ macrophages, and fibroblasts and their matrix (Figure 1C). In nonfibrotic and noninflammatory areas, SPARC expression was found in resident FSP1+ fibroblasts and was associated with collagen of the basement membrane (see Supplemental Figure S1B at http://ajp.amjpathol.org). The observation of SPARC oppositely affecting fibrosis and inflammation and the consistent detection of SPARC in both fibroblasts and inflammatory cells prompted us to investigate whether SPARC produced by leukocyte or fibroblast had distinct roles in bleomycin-induced lung damage. Cross-talk between macrophages and fibroblasts can alternatively exacerbate, suppress, or reverse fibrosis.20Wynn T.A. Integrating mechanisms of pulmonary fibrosis.J Exp Med. 2011; 208: 1339-1350Crossref PubMed Scopus (853) Google Scholar Both macrophages and fibroblasts can produce SPARC, and their distinct contribution to fibrosis could be partially investigated by reciprocal BMT between Sparc−/− and WT mice.17Sangaletti S. Di Carlo E. Gariboldi S. Miotti S. Cappetti B. Parenza M. Rumio C. Brekken R.A. Chiodoni C. Colombo M.P. Macrophage-derived SPARC bridges tumor cell-extracellular matrix interactions toward metastasis.Cancer Res. 2008; 68: 9050-9059Crossref PubMed Scopus (145) Google Scholar This approach, however, is complicated by the different origin of collagen-producing cells, which, besides resident fibroblasts, include fibrocytes21Moore B.B. Kolodsick J.E. Thannickal V.J. Cooke K. Moore T.A. Hogaboam C. Wilke C.A. Toews G.B. CCR2-mediated recruitment of fibrocytes to the alveolar space after fibrotic injury.Am J Pathol. 2005; 166: 675-684Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar and mesenchymal stem cells of bone marrow origin (BM-MSCs).22Kisseleva T. Brenner D.A. Mechanisms of fibrogenesis.Exp Biol Med (Maywood). 2008; 233: 109-122Crossref PubMed Scopus (378) Google Scholar To evaluate the contribution of bone marrow–derived collagen-producing cells in bleomycin-induced fibrosis, we designed ad hoc bone marrow chimeras, which allowed us to distinguish host- and bone marrow–derived fibroblasts and fibrocytes on the basis of CXCR4, FSP1,23Lawson W.E. Polosukhin V.V. Zoia O. Stathopoulos G.T. Han W. Plieth D. Loyd J.E. Neilson E.G. Blackwell T.S. Characterization of fibroblast-specific protein 1 in pulmonary fibrosis.Am J Respir Crit Care Med. 2005; 171: 899-907Crossref PubMed Scopus (153) Google Scholar and CD45 markers (see Supplemental Figure S2 at http://ajp.amjpathol.org). CD45.2 mice were lethally irradiated and transplanted with a CD45.1 bone marrow to distinguish donor and host cells. In such chimeras, after bleomycin treatment, numerous CXCR4+ cells localized mainly into fibrotic areas (Figure 2A), and most were CD45.1+ and therefore of donor origin (Figure 2D). IF analysis (Figure 2B) and flow cytometry (Figure 2D) revealed that among the donor population more then 85% of CXCR4+/CD45.1+ cells were leukocytes (FSP1−, α-SMA−), whereas roughly 6% of them were fibrocytes (FSP1+ or α-SMA+). FACS analysis also showed a tiny population (<2%) of CXCR4+ CD45− cells that might include BM-MSCs (Figure 2, B and D). This marginal involvement of BM-MSCs in bleomycin-treated lung was confirmed following donor CD90.2 and host CD90.1 markers on MSCs, identified by the CD146 antigen,24Tripodo C. Sangaletti S. Piccaluga P.P. Prakash S. Franco G. Borrello I. Orazi A. Colombo M.P. Pileri S.A. The bone marrow stroma in hematological neoplasms-a guilty bystander.Nat Rev Clin Oncol. 2011; 8: 456-466Crossref PubMed Scopus (40) Google Scholar in WT (Thy 1b, CD90.2)>WT (Thy 1a, CD90.1) bone marrow chimeras (see Supplemental Figure S3 at http://ajp.amjpathol.org). Donor-derived fibrocytes constituted a small fraction of the total recruited CXCR4+ cells, yet they accounted for approximately 25% (28.9% ± 5.9% in WT and 24.7% ± 8.9% in KO; Figure 2E) of the entire FSP1+ population; their relative contribution to fibrosis was evaluated according to their Sparc genotype and the ability to assemble collagen. This finding was relevant in the context of reciprocal BMT between Sparc−/− and WT mice in which collagen assembly by host or donor cells is influenced by their Sparc genotype. Indeed, resident (CXCR4−) fibroblasts and bone marrow–derived (CXCR4+) fibrocytes participated in collagen deposition in WT but not in Sparc−/− mice (Figure 2C). The results indicated that collagen-secreting cells, either resident or bone marrow derived, were impaired in ECM deposition when defective in SPARC. To confirm these data and to assess possible additional defects in proliferation and migration of Sparc−/− fibroblasts compared with WT ones, in vitro experiments were performed (see Supplemental Figure S4 at http://ajp.amjpathol.org). Dermal fibroblasts from Sparc−/− and WT mice were seeded onto polylysine-coated glasses and evaluated for collagen production by IF and Western blot25Ishida Y. Kubota H. Yamamoto A. Kitamura A. Bachinger H.P. Nagata K. Type I collagen in Hsp47-null cells is aggregated in endoplasmic reticulum and deficient in N-propeptide processing and fibrillogenesis.Mol Biol Cell. 2006; 17: 2346-2355Crossref PubMed Scopus (134) Google Scholar) (see Supplemental Figure S4A at http://ajp.amjpathol.org). Despite the similar capacity to produce collagen in WT fibroblasts, Sparc−/− fibroblasts were defective in collagen fiber assembly (see Supplemental Figure S4B at http://ajp.amjpathol.org), a process that is crucial to fibrotic disorders.26Bradshaw A.D. The role of SPARC in extracellular matrix asse" @default.
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• W2080798474 title "SPARC Oppositely Regulates Inflammation and Fibrosis in Bleomycin-Induced Lung Damage" @default.
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