Macrophage Activation on Mouse Β Autocrine-Produced

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

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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 macrophages 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 Tecnologõ«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.

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-conjugated anti-mouse IgG (Cappel, Turnhout, Belgium) was used as secondary Protein extraction and Western blot analysis Ab. An unrelated primary Ab purchased from Sigma-Aldrich was used as the control. For Western blot analysis we used a rabbit Ab against mouse Western blot analysis was performed as previously described (19). One Downloaded from iNOS (M-19; Santa Cruz Biotechnology, Santa Cruz, CA) and a mouse hundred micrograms of protein from cell lysates was loaded per lane and anti-␤-actin Ab (Sigma-Aldrich) as a control. Peroxidase-conjugated anti- separated on a 7.5% SDS-PAGE. For iNOS immunoblotting, we used a rabbit or anti-mouse IgG (Cappel) were used as secondary Abs. Blocking rabbit Ab against mouse iNOS (M-19; Santa Cruz Biotechnology) and a polyclonal Abs against TGF-␤ were obtained from Promega (Madison, mouse anti-␤-actin Ab (Sigma-Aldrich) as a control. For the analysis of WI). For analysis of TGF-␤ expression by Western blot, the same Abs TGF-␤ expression a polyclonal Ab directed to biologically active human were used. TGF-␤ (Promega) was used. Peroxidase-conjugated anti-rabbit and anti- ␣ ␤

The cDNA probes for IA- and IA- used for Northern blot analysis mouse IgG (Cappel) were used as secondary Abs. Incubations were per- http://www.jimmunol.org/ were gifts from P. Cosson (Basel Institute for Immunobiology, Basel, Swit- formed for1hatroom temperature. ECL detection was performed (Am- zerland). A rat iNOS cDNA fragment (17) was used to detect IFN-␥- and ersham Pharmacia Biotech), and the membranes were exposed to x-ray LPS-induced iNOS expression. For TNF-␣ mRNA detection we used a films (Amersham Pharmacia Biotech). cDNA probe obtained from Dr. M. Nabholz (Institut Suisse de Recherches Experimentales sur le Cancer, Epalinges, Switzerland). To study the ex- Statistical analysis pression of IL-1␤, we obtained a probe by digesting the construct pGEM1/ To calculate the statistical differences between the control and treated sam- IL-1␤ (provided by Dr. R. Wilson, Glaxo Research and Development Lim- ples (decorin or fibronectin), we used Student’s paired t test. Values of p Ͻ ited, Greenford, U.K.) with EcoRI/PstI. The IL-6 cDNA probe was a gift 0.05 or less were interpreted as significant. from Dr. S. Rohatgi (Center for Blood Research, Boston, MA). The probe for 18S rRNA was obtained as described previously (18).

Results by guest on September 28, 2021 Cell surface staining Decorin enhances IFN-␥- and LPS-dependent macrophage Cell surface staining was performed using specific Abs and was assessed activation db using cytofluorometric analysis (19) with mouse mAb anti-mouse IA (1 Having shown that decorin inhibits the M-CSF-dependent prolif- ␮g/106 cells). Adhered macrophages were collected by cell scrapping. An unrelated Ab was used as a control for nonspecificity. Cells were then eration of macrophages (14) and that the proliferative state of these washed by centrifugation through an FBS cushion. Stained cell suspensions cells modulates their activity (15), here we studied the effects of were analyzed using an EPICS XL flow cytometer (Coulter, Hialeah, FL). this proteoglycan on macrophage activation. For this purpose we Only viable cells were analyzed for surface staining, gating them based on used macrophages obtained from bone marrow cultures, since they the forward and side light scatter signals. represent a homogeneous, nontransformed population that can be ELISAs activated in vitro to induce proliferation, differentiation, or apoptosis. The secretion of inflammatory cytokines (TNF-␣, IL-1␤, and IL-6) was analyzed by ELISA using commercial murine kits following the manufac- First, we analyzed the effect of decorin on MHC class II ex- turer’s recommendations (Cytoset system; BioSource, Nivelles, Belgium). pression induced by IFN-␥, the main macrophage activator (2). In brief, 5 ϫ 105 macrophages were cultured in 24-well precoated plates in Macrophages were cultured on plates precoated with 10 ␮g/ml 0.5 ml of complete medium for 2 h. Once they attach to the plates, cells decorin or with 10 ␮g/ml BSA as a control. It is important to note were stimulated with subsaturating amounts of IFN-␥ or LPS as described, and the supernatants were recollected 24 h later and immediately used for that the indicated concentrations of decorin correspond to the con- ELISA analysis. Each sample was analyzed in triplicate, and the results are centration of the precoating solution and that we were not able to presented as the mean Ϯ SD. quantify the amount of decorin adsorbed on the plate after precoating, but other proteins under the same conditions bound Ͻ10Ð Proliferation and adhesion analysis 20%. Once attached to the plates, when cells were stimulated with Cell proliferation was analyzed by [3H]thymidine incorporation, and cell subsaturating amounts of IFN-␥ (10 U/ml), flow cytometry after adhesion to the substrate was determined by crystal violet staining as pre- 48 h showed that decorin induced a statistically significant increase viously described (14). Each sample was analyzed in triplicate, and the results are presented as the mean Ϯ SD. in MHC II protein surface expression compared with those treated only with IFN-␥ (Fig. 1, a and b). The increase in IA protein ␤ Scatchard and TGF- binding analysis surface expression correlated, as measured by Northern blotting, To analyze the binding of TGF-␤ to macrophages and the capacity of with a rise in the expression of IA-␣ and IA-␤ mRNA (Fig. 1c). decorin to modulate this process we cultured 106 cells/well in 12-well The effect of decorin on IFN-␥-induced activation was not spe- plates precoated with 10 ␮g/ml BSA or decorin. We then washed them cific for MHC II genes, since it also increased the expression of with Krebs-Ringer-HEPES (128 mM NaCl, 5 mM KCl, 5 mM Mg SO , 1.3 4 iNOS and cytokine mRNA induced by IFN-␥. Decorin slightly mM CaCl2, and 50 mM HEPES; pH 7.4). For the binding analysis we incubated cells with the indicated amounts of iodinated TGF-␤. For the increased the levels of iNOS mRNA induced by 10 U/ml IFN-␥ at Scatchard analysis, 125I-labeled TGF-␤ (100 pM) binding was competed 6 h and elongated the expression kinetics of this enzyme (Fig. 2a). 4452 DECORIN ENHANCES MACROPHAGE ACTIVATION Downloaded from http://www.jimmunol.org/

FIGURE 2. Decorin enhances IFN-␥- and LPS-dependent macrophage activation. Cells were cultured in plates precoated with BSA (10 ␮g/ml) or decorin (10 ␮g/ml) and treated with subsaturating amounts of IFN-␥ (10 FIGURE 1. Decorin increases IFN-␥-induced MHC class II expression. U/ml) or LPS (1 ng/ml) for the indicated times. The iNOS (a and d) and a, Decorin increases IFN-␥-induced MHC class II surface expression The cytokine (b and e; TNF-␣, IL-1␤, or IL-6) mRNA expression was analyzed expression of IA molecules on the cell surface was analyzed by flow cy- by Northern blotting. ELISA analysis of TNF-␣, IL-1␤, and IL-6 of the tometry. Macrophages were cultured on a 10 ␮g/ml precoated BSA or IFN-␥-treated (c) or LPS-treated (f) macrophages in the presence or the decorin surface for 24 h in the presence (dotted histogram) or absence absence of decorin were performed as indicated in Materials and Methods. p Ͻ 0.01 between BSA- and decorin-treated samples. Results are rep- by guest on September 28, 2021 ,ء ,solid line histogram) of subsaturating amounts of IFN-␥ (10 U/ml). b) Quantitation of IA surface expression using Immuno-4 software. The val- resentative of at least three independent experiments. ues shown correspond to the mean Ϯ SD of three independent experiments. -p Ͻ 0.01 between BSA- and decorin-treated samples. c, Decorin in ,ء creases IFN-␥-induced MHC class II mRNA expression. The expression of decorin elongated the expression of iNOS (Fig. 2d) and enhanced ␣ ␤ IA- and IA- mRNA was analyzed by Northern blotting. Macrophages that of IL-6 (Fig. 2e). Similar to what we observed in macrophages ␮ were cultured on a 10 g/ml precoated BSA or decorin surface and stim- treated with IFN-␥, ELISA analysis showed that decorin also in- ulated with subsaturating amounts of IFN-␥ (10 U/ml) for the indicated creases the secretion of inflammatory cytokines such as TNF-␣ or times. Quantification of the bands of interest was performed by densitom- etry, and the figure shows the mean Ϯ SD of four representative experi- IL-6 (Fig. 2f). Thus, this proteoglycan enhanced the macrophage .p Ͻ 0.01 between BSA- and decorin-treated samples. activation induced by both endogenous and exogenous activators ,ء .ments Decorin-induced adhesion does not mediate the decorin Subsaturating amounts of IFN-␥ induced low levels of TNF-␣, enhancement of macrophage activation IL-1␤, or IL-6 mRNA expression, which were only visible after Since LPS and IFN-␥ activate macrophages via distinct pathways, overexposing the film. Culture of macrophages on plates precoated we attempted to identify a possible common mechanism used by with 10 ␮g/ml decorin was enough to induce maximal expression decorin to enhance these pathways. Consistent with previous ob- of TNF-␣ and IL-1␤ mRNA (Fig. 2b). The addition of 10 U/ml servations, decorin inhibited macrophage proliferation and en- IFN-␥ did not increase this expression. While decorin alone did not hanced their adhesion (14). We also demonstrated that the prolif- induce the expression of IL-6, subsaturating amounts of IFN-␥ did erative state of macrophages could modulate the activation (Fig. 2b). ELISA analysis of the supernatants allows extends our capabilities of these cells (15). Moreover, cellular adhesion and findings, since they showed that decorin increases the secretion of integrin signaling are potent modulators of macrophage activity these inflammatory cytokines. Although decorin alone induces (20, 21). To explore the possible consequences of increased adhe- maximal mRNA expression of TNF-␣ and IL-1␤, it induced only sion and the anti-proliferative effect on the enhanced activation small amounts of secreted TNF-␣ and no IL-1␤ (Fig. 2c). How- induced by decorin, we used other components of the ECM that ever, decorin increased the secreted amounts of these cytokines in modify macrophage adhesion. response to subsaturating amounts of IFN-␥ (Fig. 2c). Macrophages cultured in plates treated with diverse ECM pro- Because decorin enhances several aspects of macrophage acti- teins showed varying degrees of adhesion. While decorin, vitro- vation induced by the endogenous activator IFN-␥, we studied the nectin, and fibronectin increased macrophage adhesion on plates effects of some components of the bacterial wall such as LPS, treated with BSA, laminin and collagen I decreased this process which could modulate diverse functions of these macrophages. We (Fig. 3). We found a negative correlation between the degree of observed that with subsaturating amounts of LPS (1 ng/ml), adhesion and proliferation (Fig. 3). Specifically, cells grown on The Journal of Immunology 4453

the expression or secretion of inflammatory cytokines in response to subsaturating amounts of IFN-␥ (Fig. 4, b and c). More differences between decorin and fibronectin were observed when we analyzed the effect of fibronectin on LPS activation of macrophages. In particular, fibronectin in response to subsaturating amounts of LPS did not induce an increase in the expression of iNOS and cytokines, but instead reduced some of them (Fig. 4, d–f).

The effects of decorin on macrophage activation are due to the sequestration of endogenous TGF-␤ Our results indicate that the antiproliferative or adhesion-inducing FIGURE 3. Negative correlation between adhesion and proliferation. 3 effects of decorin on macrophages are not sufficient to explain its Proliferation was determined by [ H]thymidine incorporation, and adhe- ability to enhance the activation of these cells. We therefore stud- sion was determined by by crystal violet staining in cells cultured in 10 ied other possible mechanisms. For example, it has been reported ␮g/ml of the indicated ECM proteins or BSA-precoated plates for 24 h. ␤ Each point was performed in triplicate, and results are represented as the that decorin binds TGF- (22, 23). However, results do not agree mean Ϯ SD of five independent experiments. The adhesive and prolifer- about whether the decorin-TGF-␤ complex has a positive or negative capacities of macrophages in untreated plates were not modified by ative effect on the interaction of TGF-␤ with its receptor on the cell the precoating of the plate with 10 ␮g/ml BSA. surface (22, 23). Due to the repressive effect of TGF-␤ on both LPS- and IFN-␥-dependent activation (24), we decided to explore Downloaded from the interaction of decorin with TGF-␤. fibronectin or decorin surfaces, to which they attached strongly, 125I-labeled rTGF-␤ bound to macrophages in a specific and proliferated less than those cultured on a BSA-precoated surface saturable manner at 4¡C (Fig. 5). In these experiments 96Ð98% of (Fig. 3). By contrast, those cultured on a surface to which they the total binding was specific, since it was blocked in the presence attached slightly, such as laminin, showed more proliferation than of a 100-fold excess of unlabeled TGF-␤. Binding was also ho- control cells. mogeneous, noncooperative, and of moderately high affinity (Fig. http://www.jimmunol.org/ In contrast to decorin, fibronectin did not increase the MHC 5a). Furthermore, TGF-␤ binding was dose-dependent and satura- ␥ class II mRNA expression induced by IFN- (Fig. 4a) or enhance ble at 250 pM (Fig. 5a). Scatchard analysis showed that macro- ␤ Ϯ phages bound TGF- with a Kd of 0.116 0.03 nM, and the number of receptors on the macrophage surface was 4793 Ϯ 813 by guest on September 28, 2021

FIGURE 4. Fibronectin does not modulate IFN-␥- or LPS-induced macrophage activation. Bone marrow-derived macrophages were cultured FIGURE 5. Decorin binds TGF-␤ and inhibits TGF-␤ binding to its in plates precoated with BSA (10 ␮g/ml) or fibronectin (10 ␮g/ml) and receptor. a, Saturating curves of 125I-labeled TGF binding to macrophages treated with subsaturating amounts of IFN-␥ (10 U/ml) or LPS (1 ng/ml) cultured on a surface precoated with 10 ␮g/ml BSA or decorin. Binding for the indicated times. MHC class II (IA-␣ and IA-␤; a), cytokine (TNF-␣, experiments were performed as described in Materials and Methods. Each IL-1␤, or IL-6; b and e), or iNOS (d) mRNA expression were analyzed by point was determined in triplicate and is presented as the mean Ϯ SD. b, Northern blotting. Results are representative of four independent experi- Scatchard analysis of 125I-labeled TGF-␤ in the presence of increasing ments. ELISA analysis of TNF-␣, IL-1␤, and IL-6 of the IFN-␥-treated (c) concentrations of unlabeled TGF-␤ in macrophages cultured on a surface or LPS-treated (f) macrophages in the presence or the absence of decorin precoated with 10 ␮g/ml BSA or decorin. Each measurement was made in p Ͻ 0.01 between triplicate and is presented as the mean Ϯ SD. The Scatchard analysis shows ,ء .was performed as indicated in Materials and Methods BSA- and fibronectin-treated samples. one representative of three independent experiments. 4454 DECORIN ENHANCES MACROPHAGE ACTIVATION receptors/cell, similar to that reported for other cell types (reviewed in Ref. 25) (Fig. 5b). However, culture of cells on a plate precoated with 10 ␮g/ml decorin abolished the binding of TGF-␤ to macrophages even at the highest 125I-labeled TGF-␤ level tested (500 pM; Fig. 5). Collectively, these binding experiments showed that TGF-␤ binds to the macrophage surface and that decorin inhibits binding of the cytokine, preventing it from interacting with its receptor in macrophages. Next, we explored whether decorin could suppress the inhibitory capability of exogenous TGF-␤ to block macrophage activation. Treatment of macrophages with 10 ng/ml TGF-␤ was sufficient to completely block the expression of IFN-␥-induced MHC class II mRNA. However, when macrophages were cultured in the presence of decorin, TGF-␤ did not block MHC II expression (Fig. 6a). Similar results were observed when we analyzed the effects of TGF-␤ and decorin on the expression of iNOS or IL-6 mRNA induced by LPS (Fig. 6b). Our results show that decorin blocked the binding of TGF-␤ to the corresponding receptor, thereby reversing its inhibitory effects Downloaded from on macrophage activation. However, these results do not explain the increase in macrophage activation produced by decorin in the absence of exogenous TGF-␤. However, an explanation was provided when IFN-␥- or LPS-activated macrophages were treated with blocking Abs against TGF-␤. Like decorin, under these con-

ditions anti-TGF-␤ Abs increased iNOS expression induced by http://www.jimmunol.org/ subsaturating amounts of IFN-␥ (Fig. 7a) or LPS (Fig. 7b). Anti- TGF-␤ and decorin had no effect on iNOS expression in the absence of macrophage activators. FIGURE 7. The effects of decorin on macrophage activation are due to sequestration of endogenous TGF-␤. Cells were cultured in plates precoated with 10 ␮g/ml BSA or decorin. After attachment to the plate, BSA- adhered cells were treated with 1 ␮g/ml anti-TGF-␤ polyclonal Abs for 30 min or were left untreated. Then BSA- and decorin-precoated plates were

stimulated with subsaturating amounts of IFN-␥ (a) or LPS (b)for6hor by guest on September 28, 2021 were left unstimulated, and the expression of iNOS was analyzed by West- ern blot analysis. Each experiment was performed twice. c, Expression of TGF-␤ after treatment with subsaturating amounts of IFN-␥ (10 U/ml) or LPS (1 ng/ml) for 6 h was analyzed by Western blotting as indicated in Materials and Methods. Results are representative of three independent experiments.

Moreover, Western blot analyses showed that at subsaturating amounts, both IFN-␥ and LPS induced the expression of TGF-␤ in macrophages (Fig. 7c). Since decorin did not modify this expression, the blocking effect of this proteoglycan was due to TGF-␤ sequestration and inhibition of TGF-␤ binding to its receptor.

Discussion Under a range of external stimuli, macrophages proliferate, become activated and carry out their function, remain quiescent, or FIGURE 6. Decorin blocks the immunosuppressive effect of TGF-␤ on die through apoptosis. Like other immune cells, macrophages are macrophage activation. a, TGF-␤ suppresses IFN-␥-induced MHC II ex- produced in large excess, and only the few cells required to de- pression, and this effect is reversed by decorin. Macrophages were cultured velop a functional activity survive. We found that IFN-␥ not only on plates precoated with BSA or decorin. Once attached, cells were stim- activated macrophages, but also prevented the induction of apo- ulated with 1 ng/ml TGF-␤ for 30 min and then activated with subsatu- ptosis through the expression of the cdk inhibitor p21waf-1 and the ␥ rating amounts of IFN- for 6, 12, and 24 h. The expression of IA mRNAs arrest of cell cycling at the G /S boundary (3). was analyzed by Northern blot. b, TGF-␤ blocks LPS-induced iNOS and 1 In a previous study we observed that decorin, like IFN-␥, blocks IL-6 expression, and this effect is inhibited by decorin. Macrophages were ␮ macrophage proliferation and protects against apoptosis through cultured in plates precoated with 10 g/ml BSA or decorin. They were then waf1 stimulated with 1 ng/ml TGF-␤ for 30 min and activated with subsaturating the induction of p21 (14). In the present study we found that amounts of LPS for the indicated times. The expression of iNOS and IL-6 decorin enhanced both IFN-␥- and LPS-mediated activation. This mRNAs was analyzed by Northern blot. The results in a and b are repre- enhancing effect was originally linked to the capacity of decorin to sentative of at least three independent experiments. increase cell adhesion on the basis that fibronectin, another ECM The Journal of Immunology 4455 protein that induces cell adhesion, also inhibits macrophage pro- macrophages reach these loci, where they remain until inflamma- liferation. However, we observed that decorin increased the acti- tion disappears (39), i.e., for as long as stimulated Th1 cells pro- vation mediated by both LPS and IFN-␥, whereas fibronectin duce IFN-␥. In the late phases of inflammation, macrophages elim- did not. inate nonself structures, remove all debris (including apoptotic Because decorin-induced adhesion did not seem to be a suffi- bodies), and remodel impaired tissues. However, during chronic cient mechanism to increase macrophage activation, we analyzed inflammation, such as rheumatoid arthritis, macrophages play a the repressive effect of this proteoglycan on TGF-␤. This cytokine key role in the pathogenesis (40). In these situations the persistence is produced by macrophages in an autocrine manner, and it down- of these phagocytic cells may be related to the presence of mole- regulates activation (26). TGF-␤ antagonizes IFN-␥-driven pro- cules that block their deactivation. Several soluble mediators, such ␤ cesses of macrophage activation, such as the production of H2O2, as TGF- (28Ð30), IL-10 (30, 41), adenosine (19), etc., block mac- NO, the up-regulation of iNOS, the release of TNF-␣, or the IFN- rophage activation. Macrophages are restrained from tissue-dam- ␥-induced death of intracellular microorganisms (27Ð30). TGF-␤ aging activation by CD200R (a myeloid-specific receptor on the also shows an inhibitory effect on IFN-␥-induced MHC class II phagocytes) when it engages on other cells the glycoprotein genes and is mediated by a conserved proximal promoter element CD200 (42, 43). Depending on the balance between activators and (31). The repressive effect of TGF-␤ on IFN-␥ is based on cross- inhibitors, macrophages remain at the inflammatory loci and re- talk between the molecules involved in signal transduction path- lease enzymes or cytokines that could be deleterious for the artic- ways. The TGF-␤/SMAD signaling cascades are inhibited by IFN- ulation (44). In this context, decorin or other molecules of the ␥/STAT pathways and vice versa (32, 33). In addition, TGF-␤ ECM may contribute to the pathogenesis of chronic inflammation inhibits LPS-induced activation of macrophages. This cytokine in- by blocking inhibitors. In this regard, in an animal model of ex- Downloaded from hibits LPS-induced iNOS expression (34) or reduces the expres- perimental autoimmune encephalomyelitis, systemic administra- sion of proinflammatory cytokines during septic shock (35). tion of Abs specific for TGF-␤ identified a role for endogenous Our results also show that treatment of macrophages with TGF-␤ in suppression of the disease (45). decorin alone is sufficient to induce the mRNA expression of some cytokines, such as TNF-␣ and IL-1␤, but it is not able to induce Acknowledgments their secretion. This indicates that although treatment with decorin We give special thanks to Dr. E. Ruoslahti (The Burnham Institute, La http://www.jimmunol.org/ alone could modulate gene transcription, it does not regulate the Jolla, CA) for the gift of the purified decorin used in this study. We also post-transcriptional mechanisms involved in cytokine secretion. thank Dr. J. Gascon (Hospital Clinic, Barcelona, Spain) and Puleva Biotech Increased expression and secretion of these inflammatory cyto- (Granada, Spain) for their help with some reagents. We thank Tanya Yates kines induced by decorin were only observed in the presence of for editorial help. macrophage activators such as LPS or IFN-␥. Here we found that macrophages bound TGF-␤ with an affinity References and number of binding sites per cell similar to those observed in 1. Celada, A., and C. F. Nathan. 1994. Macrophage activation revisited. Immunol. ␤ Today 15:100. other cell types (25). Decorin also blocked the binding of TGF- 2. Boehm, U., T. Klamp, and M. Groot. 1997. Cellular responses to interferon ␥. to macrophages, which could be due to binding of decorin to Annu. Rev. Immunol. 15:749. by guest on September 28, 2021 TGF-␤ and inhibition of the interaction with the cell surface re- 3. Xaus, J., M. Cardo, A. F. Valledor, C. Soler, J. Lloberas, and A. Celada. 1999. 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