Macrophage Activation on Mouse Β Autocrine-Produced
Total Page:16
File Type:pdf, Size:1020Kb
Decorin Reverses the Repressive Effect of Autocrine-Produced TGF- β on Mouse Macrophage Activation This information is current as Mònica Comalada, Marina Cardó, Jordi Xaus, Annabel F. of September 28, 2021. Valledor, Jorge Lloberas, Francesc Ventura and Antonio Celada J Immunol 2003; 170:4450-4456; ; doi: 10.4049/jimmunol.170.9.4450 http://www.jimmunol.org/content/170/9/4450 Downloaded from References This article cites 45 articles, 26 of which you can access for free at: http://www.jimmunol.org/content/170/9/4450.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 28, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Decorin Reverses the Repressive Effect of Autocrine-Produced TGF- on Mouse Macrophage Activation1 Mo`nica Comalada,2* Marina Cardo´,2,3* Jordi Xaus,2* Annabel F. Valledor,* Jorge Lloberas,* Francesc Ventura,† and Antonio Celada4* Several cytokines or growth factors induce macrophages to proliferate, become activated, differentiate, or die through apoptosis. Like the major macrophage activator IFN-␥, the extracellular matrix protein decorin inhibits proliferation and protects macro- phages from the induction of apoptosis. Decorin enhances the IFN-␥-induced expression of the IA␣ and IA MHC class II genes. Moreover, it increases the IFN-␥- or LPS-induced expression of inducible NO synthase, TNF-␣, IL-1, and IL-6 genes and the secretion of these cytokines. Using a number of extracellular matrix proteins, we found a negative correlation between adhesion and proliferation. However, the effects of decorin on macrophage activation do not seem to be mediated through its effect on adhesion or proliferation. Instead, this proteoglycan abolishes the binding of TGF- to macrophages, as shown by Scatchard Downloaded from ϳ ؎  125 analysis of I-labeled TGF- , which, in the absence of decorin, showed a Kd of 0.11 0.03 nM and 5000 receptors/cell. This was confirmed when we treated macrophages with Abs to block the endogenously produced TGF-, which enhanced macrophage activation in a way similar to decorin. The increase in activation mediated by decorin demonstrates that macrophages are under negative regulation that can be reversed by proteins of the extracellular matrix. The Journal of Immunology, 2003, 170: 4450–4456. http://www.jimmunol.org/ acrophages play a key role in the immune response. leucine-rich proteoglycans, which are found in the extracellular These phagocytic cells are produced in the bone mar- matrix (ECM)5 of a variety of tissues (8). Although the biological M row and transported in blood to distinct tissues. Most role of these molecules is unclear, several observations indicate macrophages die through apoptosis; however, in the presence of that decorin and perhaps other proteoglycans regulate the remod- certain cytokines or growth factors, they proliferate, differentiate eling of connective tissue. In particular, binding studies in vitro into several cell types (Kupffer cells, Langerhans, microglia, etc.), have shown that decorin interacts with several types of collagen, or become activated to develop their functions. At the inflamma- and it is believed to be a key regulator of collagen fibrillogenesis tory loci, macrophage phagocyte bacteria, remove cell debris, re- (9). This proteoglycan may also affect the production of other lease several mediators, present Ags to T lymphocytes, and con- ECM components (10, 11). Additionally, decorin modulates the in- by guest on September 28, 2021 tribute to the resolution of inflammation (1). teractions of matrix molecules such as fibronectin with cells (12, 13). IFN-␥, which is released by activated T lymphocytes or NK Decorin, like IFN-␥, inhibits the proliferation of macrophages and cells, is the most potent activator of macrophages and induces the enhances cell survival through the expression of p27Kip1 and p21waf-1, expression of Ͼ300 genes (2). We found that this cytokine also respectively (14). Since we previously found that activation by IFN-␥ blocks macrophage proliferation and protects against apoptosis (3). or LPS inhibits the proliferation of these phagocytic cells (15), here This protection allows macrophages to survive at the inflammatory we studied the effect of decorin on macrophage activation. We used loci when IFN-␥ is present and explains the key role that T lym- primary cultures of bone marrow-derived macrophages, a homoge- phocytes play in delayed hypersensitivity (4, 5). neous cell population that responds to physiological proliferative or At the inflammatory loci, proteoglycans are secreted by mono- activating stimuli (16). Decorin enhances both LPS- and IFN-␥-in- cytes and macrophages (6, 7) and modulate the immune response. duced activation, as shown by the capacity to increase MHC class II, Decorin and other related molecules form a family called small inducible NO synthase (iNOS), and cytokine expression. The effect of this proteoglycan is explained by its ability to block the binding of autocrine-produced TGF- on the surface of macrophages. *Group of Macrophage Biology, Biomedical Research Institute of Barcelona-Sci- ences Park, and †Departament de Ciencias Fisiologicas II, Facultat d’Odontologia, Campus de Bellvitge, Universitat de Barcelona, Barcelona, Spain Materials and Methods Received for publication October 10, 2002. Accepted for publication February Reagents 24, 2003. Recombinant purified decorin was a gift from Dr. E. Ruoslahti (The Burn- The costs of publication of this article were defrayed in part by the payment of page ham Institute, La Jolla, CA). LPS, BSA, collagen I, vitronectin, laminin, charges. This article must therefore be hereby marked advertisement in accordance and fibronectin were obtained from Sigma-Aldrich (St. Louis, MO). with 18 U.S.C. Section 1734 solely to indicate this fact. [3H]thymidine, TGF-, and 125I-labeled TGF- were purchased from Am- 1 This work was supported by grants from the Ministerio de Ciencia y Tecnologı´a ersham Pharmacia Biotech (Uppsala, Sweden). IFN-␥ was a gift from Ge- (BMC2001-3040 to A.C. and BMC2002-00737 to F.V.). M.C. was the recipient of a nentech (South San Francisco, CA). All other products were of the highest fellowship from the Fundacio´August Pi i Sunyer. analytical grade available and were purchased from Sigma-Aldrich. Deion- 2 M.C., M.C., and J.X. contributed equally to this work. ized water that had been further purified with a Millipore Milli-Q system 3 Current address: Division of Genito-Urinary Oncology, M. D. Anderson Medical (Bedford, MA) was used. Center, Houston, TX 78957. 4 Address correspondence and reprint requests to Dr. Antonio Celada, Research In- stitute of Biomedicine of Barcelona-Sciences Park, Josep Samitier 1-5, 08028 Bar- 5 Abbreviations used in this paper: ECM, extracellular matrix; iNOS, inducible NO celona, Spain. E-mail address: [email protected] synthase. Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 The Journal of Immunology 4451 Cell culture with increasing amounts of cold TGF-. Cells were incubated on a rotating platform for3hat4°C. They were then washed and cross-linked for 15 min Bone marrow-derived macrophages were isolated from 6-wk-old BALB/c at 4°C with 0.5 ml of dioctyl sodium sulfosuccinate solution (6 g/ml in mice (Charles River Laboratories, Wilmington, MA) as previously de- Krebs-Ringer-HEPES). After two washes with 0.25 M sucrose, 10 mM scribed (16). Cells were cultured in plastic tissue culture dishes (150 mm) Tris-HCl, and 1 mM EDTA, proteins were solubilized with 200 l of 0.5% in 40 ml of DMEM containing 20% FBS and 30% L cell-conditioned Triton-Tris-HCl-EDTA and protease inhibitors for 40 min at 4°C. The medium as a source of M-CSF. They were then incubated at 37°Cina supernatants were then transferred to test tubes, boiled for 1 min, and humidified 5% CO2 atmosphere. After 7 days of culture, a homogeneous counted using a Packard gamma counter (Downers Grove, IL). Each point Ͼ ϩ population of adherent macrophages was obtained ( 99% Mac-1 ). was determined in triplicate, and the results are expressed as the In some circumstances cells were cultured on a precoated plate using mean Ϯ SD. distinct components of the ECM or BSA as a control. For precoating, the plates were incubated overnight at 4°C with a PBS solution of the indicated Northern blot analysis concentration of each ECM component. After coating, the plates were blocked with PBS/10 g/ml BSA for1hat37°C, the blocking solution Northern blot analysis was performed as previously described (14) using was then removed, and the cells were cultured with normal complete 20 g of total cellular RNA/lane. To check for differences in RNA loading, medium. we analyzed the expression of the 18S rRNA transcripts. All probes were labeled with [␣-32P]dCTP (ICN Pharmaceuticals, Costa Mesa, CA) with Abs and constructs the oligolabeling kit method (Amersham Pharmacia Biotech). The bands of interest were quantified with a Molecular Analyst system (Bio-Rad, For analysis of IA surface expression by flow cytometry, we used purified Richmond, CA). anti-mouse IAd,b mAbs (BD PharMingen, San Diego, CA). FITC-conju- gated anti-mouse IgG (Cappel, Turnhout, Belgium) was used as secondary Protein extraction and Western blot analysis Ab.