Decorin Reverses the Repressive Effect of Autocrine-Produced TGF-β on Mouse Macrophage Activation

Mònica Comalada, Marina Cardó, Jordi Xaus, Annabel F. Valledor, Jorge Lloberas, Francesc Ventura and Antonio Celada
J Immunol May 1, 2003, 170 (9) 4450-4456; DOI: https://doi.org/10.4049/jimmunol.170.9.4450

Abstract

Several cytokines or growth factors induce macrophages to proliferate, become activated, differentiate, or die through apoptosis. Like the major macrophage activator IFN-γ, the extracellular matrix protein decorin inhibits proliferation and protects macrophages from the induction of apoptosis. Decorin enhances the IFN-γ-induced expression of the IAα and IAβ MHC class II genes. Moreover, it increases the IFN-γ- or LPS-induced expression of inducible NO synthase, TNF-α, IL-1β, 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-β to macrophages, as shown by Scatchard analysis of 125I-labeled TGF-β, 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-β, 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.

Macrophages play a key role in the immune response. These phagocytic cells are produced in the bone marrow and transported in blood to distinct tissues. Most macrophages die through apoptosis; however, in the presence of certain cytokines or growth factors, they proliferate, differentiate into several cell types (Kupffer cells, Langerhans, microglia, etc.), or become activated to develop their functions. At the inflammatory loci, macrophage phagocyte bacteria, remove cell debris, release several mediators, present Ags to T lymphocytes, and contribute to the resolution of inflammation (1).

IFN-γ, which is released by activated T lymphocytes or NK cells, is the most potent activator of macrophages and induces the expression of >300 genes (2). We found that this cytokine also blocks macrophage proliferation and protects against apoptosis (3). This protection allows macrophages to survive at the inflammatory loci when IFN-γ is present and explains the key role that T lymphocytes play in delayed hypersensitivity (4, 5).

At the inflammatory loci, proteoglycans are secreted by monocytes and macrophages (6, 7) and modulate the immune response. Decorin and other related molecules form a family called small leucine-rich proteoglycans, which are found in the extracellular matrix (ECM)5 of a variety of tissues (8). Although the biological role of these molecules is unclear, several observations indicate that decorin and perhaps other proteoglycans regulate the remodeling of connective tissue. In particular, binding studies in vitro have shown that decorin interacts with several types of collagen, and it is believed to be a key regulator of collagen fibrillogenesis (9). This proteoglycan may also affect the production of other ECM components (10, 11). Additionally, decorin modulates the interactions of matrix molecules such as fibronectin with cells (12, 13).

Decorin, like IFN-γ, inhibits the proliferation of macrophages and enhances cell survival through the expression of p27Kip1 and p21waf-1, respectively (14). Since we previously found that activation by IFN-γ or LPS inhibits the proliferation of these phagocytic cells (15), here we studied the effect of decorin on macrophage activation. We used primary cultures of bone marrow-derived macrophages, a homogeneous cell population that responds to physiological proliferative or activating stimuli (16). Decorin enhances both LPS- and IFN-γ-induced activation, as shown by the capacity to increase MHC class II, 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-β on the surface of macrophages.

Materials and Methods

Reagents

Recombinant purified decorin was a gift from Dr. E. Ruoslahti (The Burnham Institute, La Jolla, CA). LPS, BSA, collagen I, vitronectin, laminin, and fibronectin were obtained from Sigma-Aldrich (St. Louis, MO). [3H]thymidine, TGF-β, and 125I-labeled TGF-β were purchased from Amersham Pharmacia Biotech (Uppsala, Sweden). IFN-γ was a gift from Genentech (South San Francisco, CA). All other products were of the highest analytical grade available and were purchased from Sigma-Aldrich. Deionized water that had been further purified with a Millipore Milli-Q system (Bedford, MA) was used.

Cell culture

Bone marrow-derived macrophages were isolated from 6-wk-old BALB/c mice (Charles River Laboratories, Wilmington, MA) as previously described (16). Cells were cultured in plastic tissue culture dishes (150 mm) in 40 ml of DMEM containing 20% FBS and 30% L cell-conditioned medium as a source of M-CSF. They were then incubated at 37°C in a humidified 5% CO2 atmosphere. After 7 days of culture, a homogeneous population of adherent macrophages was obtained (>99% Mac-1+).

In some circumstances cells were cultured on a precoated plate using 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 concentration of each ECM component. After coating, the plates were blocked with PBS/10 μg/ml BSA for 1 h at 37°C, the blocking solution was then removed, and the cells were cultured with normal complete medium.

Abs and constructs

For analysis of IA surface expression by flow cytometry, we used purified anti-mouse IAd,b mAbs (BD PharMingen, San Diego, CA). FITC-conjugated anti-mouse IgG (Cappel, Turnhout, Belgium) was used as secondary 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 iNOS (M-19; Santa Cruz Biotechnology, Santa Cruz, CA) and a mouse anti-β-actin Ab (Sigma-Aldrich) as a control. Peroxidase-conjugated anti-rabbit or anti-mouse IgG (Cappel) were used as secondary Abs. Blocking polyclonal Abs against TGF-β were obtained from Promega (Madison, WI). For analysis of TGF-β expression by Western blot, the same Abs were used.

The cDNA probes for IA-α and IA-β used for Northern blot analysis were gifts from P. Cosson (Basel Institute for Immunobiology, Basel, Switzerland). A rat iNOS cDNA fragment (17) was used to detect IFN-γ- and LPS-induced iNOS expression. For TNF-α mRNA detection we used a cDNA probe obtained from Dr. M. Nabholz (Institut Suisse de Recherches Experimentales sur le Cancer, Epalinges, Switzerland). To study the expression of IL-1β, we obtained a probe by digesting the construct pGEM1/IL-1β (provided by Dr. R. Wilson, Glaxo Research and Development Limited, Greenford, U.K.) with EcoRI/PstI. The IL-6 cDNA probe was a gift from Dr. S. Rohatgi (Center for Blood Research, Boston, MA). The probe for 18S rRNA was obtained as described previously (18).

Cell surface staining

Cell surface staining was performed using specific Abs and was assessed using cytofluorometric analysis (19) with mouse mAb anti-mouse IAdb (1 μg/106 cells). Adhered macrophages were collected by cell scrapping. An unrelated Ab was used as a control for nonspecificity. Cells were then washed by centrifugation through an FBS cushion. Stained cell suspensions were analyzed using an EPICS XL flow cytometer (Coulter, Hialeah, FL). Only viable cells were analyzed for surface staining, gating them based on the forward and side light scatter signals.

ELISAs

The secretion of inflammatory cytokines (TNF-α, IL-1β, and IL-6) was analyzed by ELISA using commercial murine kits following the manufacturer’s recommendations (Cytoset system; BioSource, Nivelles, Belgium). In brief, 5 × 105 macrophages were cultured in 24-well precoated plates in 0.5 ml of complete medium for 2 h. Once they attach to the plates, cells were stimulated with subsaturating amounts of IFN-γ or LPS as described, and the supernatants were recollected 24 h later and immediately used for ELISA analysis. Each sample was analyzed in triplicate, and the results are presented as the mean ± SD.

Proliferation and adhesion analysis

Cell proliferation was analyzed by [3H]thymidine incorporation, and cell adhesion to the substrate was determined by crystal violet staining as previously described (14). Each sample was analyzed in triplicate, and the results are presented as the mean ± SD.

Scatchard and TGF-β binding analysis

To analyze the binding of TGF-β to macrophages and the capacity of decorin to modulate this process we cultured 106 cells/well in 12-well plates precoated with 10 μg/ml BSA or decorin. We then washed them with Krebs-Ringer-HEPES (128 mM NaCl, 5 mM KCl, 5 mM Mg SO4, 1.3 mM CaCl2, and 50 mM HEPES; pH 7.4). For the binding analysis we incubated cells with the indicated amounts of iodinated TGF-β. For the Scatchard analysis, 125I-labeled TGF-β (100 pM) binding was competed with increasing amounts of cold TGF-β. Cells were incubated on a rotating platform for 3 h at 4°C. They were then washed and cross-linked for 15 min at 4°C with 0.5 ml of dioctyl sodium sulfosuccinate solution (6 μg/ml in Krebs-Ringer-HEPES). After two washes with 0.25 M sucrose, 10 mM Tris-HCl, and 1 mM EDTA, proteins were solubilized with 200 μl of 0.5% Triton-Tris-HCl-EDTA and protease inhibitors for 40 min at 4°C. The supernatants were then transferred to test tubes, boiled for 1 min, and counted using a Packard gamma counter (Downers Grove, IL). Each point was determined in triplicate, and the results are expressed as the mean ± SD.

Northern blot analysis

Northern blot analysis was performed as previously described (14) using 20 μg of total cellular RNA/lane. To check for differences in RNA loading, we analyzed the expression of the 18S rRNA transcripts. All probes were labeled with [α-32P]dCTP (ICN Pharmaceuticals, Costa Mesa, CA) with the oligolabeling kit method (Amersham Pharmacia Biotech). The bands of interest were quantified with a Molecular Analyst system (Bio-Rad, Richmond, CA).

Protein extraction and Western blot analysis

Western blot analysis was performed as previously described (19). One hundred micrograms of protein from cell lysates was loaded per lane and separated on a 7.5% SDS-PAGE. For iNOS immunoblotting, we used a rabbit Ab against mouse iNOS (M-19; Santa Cruz Biotechnology) and a mouse anti-β-actin Ab (Sigma-Aldrich) as a control. For the analysis of TGF-β expression a polyclonal Ab directed to biologically active human TGF-β (Promega) was used. Peroxidase-conjugated anti-rabbit and anti-mouse IgG (Cappel) were used as secondary Abs. Incubations were performed for 1 h at room temperature. ECL detection was performed (Amersham Pharmacia Biotech), and the membranes were exposed to x-ray films (Amersham Pharmacia Biotech).

Statistical analysis

To calculate the statistical differences between the control and treated samples (decorin or fibronectin), we used Student’s paired t test. Values of p < 0.05 or less were interpreted as significant.

Results

Decorin enhances IFN-γ- and LPS-dependent macrophage activation

Having shown that decorin inhibits the M-CSF-dependent proliferation of macrophages (14) and that the proliferative state of these cells modulates their activity (15), here we studied the effects of this proteoglycan on macrophage activation. For this purpose we used macrophages obtained from bone marrow cultures, since they represent a homogeneous, nontransformed population that can be activated in vitro to induce proliferation, differentiation, or apoptosis.

First, we analyzed the effect of decorin on MHC class II expression induced by IFN-γ, the main macrophage activator (2). Macrophages were cultured on plates precoated with 10 μg/ml decorin or with 10 μg/ml BSA as a control. It is important to note that the indicated concentrations of decorin correspond to the concentration of the precoating solution and that we were not able to quantify the amount of decorin adsorbed on the plate after precoating, but other proteins under the same conditions bound <10–20%. Once attached to the plates, when cells were stimulated with subsaturating amounts of IFN-γ (10 U/ml), flow cytometry after 48 h showed that decorin induced a statistically significant increase in MHC II protein surface expression compared with those treated only with IFN-γ (Fig. 1⇓, a and b). The increase in IA protein surface expression correlated, as measured by Northern blotting, with a rise in the expression of IA-α and IA-β mRNA (Fig. 1⇓c).

FIGURE 1.
FIGURE 1.

Decorin increases IFN-γ-induced MHC class II expression. a, Decorin increases IFN-γ-induced MHC class II surface expression The expression of IA molecules on the cell surface was analyzed by flow cytometry. Macrophages were cultured on a 10 μg/ml precoated BSA or decorin surface for 24 h in the presence (dotted histogram) or absence (solid line histogram) of subsaturating amounts of IFN-γ (10 U/ml). b, Quantitation of IA surface expression using Immuno-4 software. The values shown correspond to the mean ± SD of three independent experiments. ∗, p < 0.01 between BSA- and decorin-treated samples. c, Decorin increases IFN-γ-induced MHC class II mRNA expression. The expression of IA-α and IA-β mRNA was analyzed by Northern blotting. Macrophages were cultured on a 10 μg/ml precoated BSA or decorin surface and stimulated with subsaturating amounts of IFN-γ (10 U/ml) for the indicated times. Quantification of the bands of interest was performed by densitometry, and the figure shows the mean ± SD of four representative experiments. ∗, p < 0.01 between BSA- and decorin-treated samples.

The effect of decorin on IFN-γ-induced activation was not specific for MHC II genes, since it also increased the expression of iNOS and cytokine mRNA induced by IFN-γ. Decorin slightly increased the levels of iNOS mRNA induced by 10 U/ml IFN-γ at 6 h and elongated the expression kinetics of this enzyme (Fig. 2⇓a). Subsaturating amounts of IFN-γ induced low levels of TNF-α, IL-1β, or IL-6 mRNA expression, which were only visible after overexposing the film. Culture of macrophages on plates precoated with 10 μg/ml decorin was enough to induce maximal expression of TNF-α and IL-1β mRNA (Fig. 2⇓b). The addition of 10 U/ml IFN-γ did not increase this expression. While decorin alone did not induce the expression of IL-6, subsaturating amounts of IFN-γ did (Fig. 2⇓b). ELISA analysis of the supernatants allows extends our findings, since they showed that decorin increases the secretion of these inflammatory cytokines. Although decorin alone induces maximal mRNA expression of TNF-α and IL-1β, it induced only small amounts of secreted TNF-α and no IL-1β (Fig. 2⇓c). However, decorin increased the secreted amounts of these cytokines in response to subsaturating amounts of IFN-γ (Fig. 2⇓c).

FIGURE 2.
FIGURE 2.

Decorin enhances IFN-γ- and LPS-dependent macrophage activation. Cells were cultured in plates precoated with BSA (10 μg/ml) or decorin (10 μg/ml) and treated with subsaturating amounts of IFN-γ (10 U/ml) or LPS (1 ng/ml) for the indicated times. The iNOS (a and d) and cytokine (b and e; TNF-α, IL-1β, or IL-6) mRNA expression was analyzed by Northern blotting. ELISA analysis of TNF-α, IL-1β, and IL-6 of the IFN-γ-treated (c) or LPS-treated (f) macrophages in the presence or the absence of decorin were performed as indicated in Materials and Methods. ∗, p < 0.01 between BSA- and decorin-treated samples. Results are representative of at least three independent experiments.

Because decorin enhances several aspects of macrophage activation induced by the endogenous activator IFN-γ, we studied the effects of some components of the bacterial wall such as LPS, which could modulate diverse functions of these macrophages. We observed that with subsaturating amounts of LPS (1 ng/ml), decorin elongated the expression of iNOS (Fig. 2⇑d) and enhanced that of IL-6 (Fig. 2⇑e). Similar to what we observed in macrophages treated with IFN-γ, ELISA analysis showed that decorin also increases the secretion of inflammatory cytokines such as TNF-α or IL-6 (Fig. 2⇑f). Thus, this proteoglycan enhanced the macrophage activation induced by both endogenous and exogenous activators.

Decorin-induced adhesion does not mediate the decorin enhancement of macrophage activation

Since LPS and IFN-γ activate macrophages via distinct pathways, we attempted to identify a possible common mechanism used by decorin to enhance these pathways. Consistent with previous observations, decorin inhibited macrophage proliferation and enhanced their adhesion (14). We also demonstrated that the proliferative state of macrophages could modulate the activation capabilities of these cells (15). Moreover, cellular adhesion and integrin signaling are potent modulators of macrophage activity (20, 21). To explore the possible consequences of increased adhesion and the anti-proliferative effect on the enhanced activation induced by decorin, we used other components of the ECM that modify macrophage adhesion.

Macrophages cultured in plates treated with diverse ECM proteins showed varying degrees of adhesion. While decorin, vitronectin, and fibronectin increased macrophage adhesion on plates treated with BSA, laminin and collagen I decreased this process (Fig. 3⇓). We found a negative correlation between the degree of adhesion and proliferation (Fig. 3⇓). Specifically, cells grown on fibronectin or decorin surfaces, to which they attached strongly, proliferated less than those cultured on a BSA-precoated surface (Fig. 3⇓). By contrast, those cultured on a surface to which they attached slightly, such as laminin, showed more proliferation than control cells.

FIGURE 3.
FIGURE 3.

Negative correlation between adhesion and proliferation. Proliferation was determined by [3H]thymidine incorporation, and adhesion was determined by by crystal violet staining in cells cultured in 10 μg/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 mean ± SD of five independent experiments. The adhesive and proliferative capacities of macrophages in untreated plates were not modified by the precoating of the plate with 10 μg/ml BSA.

In contrast to decorin, fibronectin did not increase the MHC class II mRNA expression induced by IFN-γ (Fig. 4⇓a) or enhance the expression or secretion of inflammatory cytokines in response to subsaturating amounts of IFN-γ (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).

FIGURE 4.
FIGURE 4.

Fibronectin does not modulate IFN-γ- or LPS-induced macrophage activation. Bone marrow-derived macrophages were cultured in plates precoated with BSA (10 μg/ml) or fibronectin (10 μg/ml) and treated with subsaturating amounts of IFN-γ (10 U/ml) or LPS (1 ng/ml) for the indicated times. MHC class II (IA-α and IA-β; a), cytokine (TNF-α, IL-1β, or IL-6; b and e), or iNOS (d) mRNA expression were analyzed by Northern blotting. Results are representative of four independent experiments. ELISA analysis of TNF-α, IL-1β, and IL-6 of the IFN-γ-treated (c) or LPS-treated (f) macrophages in the presence or the absence of decorin was performed as indicated in Materials and Methods. ∗, p < 0.01 between BSA- and fibronectin-treated samples.

The effects of decorin on macrophage activation are due to the sequestration of endogenous TGF-β

Our results indicate that the antiproliferative or adhesion-inducing effects of decorin on macrophages are not sufficient to explain its ability to enhance the activation of these cells. We therefore studied other possible mechanisms. For example, it has been reported that decorin binds TGF-β (22, 23). However, results do not agree about whether the decorin-TGF-β complex has a positive or negative effect on the interaction of TGF-β with its receptor on the cell surface (22, 23). Due to the repressive effect of TGF-β on both LPS- and IFN-γ-dependent activation (24), we decided to explore the interaction of decorin with TGF-β.

125I-labeled rTGF-β bound to macrophages in a specific and saturable manner at 4°C (Fig. 5⇓). In these experiments 96–98% of the total binding was specific, since it was blocked in the presence of a 100-fold excess of unlabeled TGF-β. Binding was also homogeneous, noncooperative, and of moderately high affinity (Fig. 5⇓a). Furthermore, TGF-β binding was dose-dependent and saturable at 250 pM (Fig. 5⇓a). Scatchard analysis showed that macrophages bound TGF-β with a Kd of 0.116 ± 0.03 nM, and the number of receptors on the macrophage surface was 4793 ± 813 receptors/cell, similar to that reported for other cell types (reviewed in Ref. 25) (Fig. 5⇓b). However, culture of cells on a plate precoated with 10 μg/ml decorin abolished the binding of TGF-β to macrophages even at the highest 125I-labeled TGF-β level tested (500 pM; Fig. 5⇓). Collectively, these binding experiments showed that TGF-β binds to the macrophage surface and that decorin inhibits binding of the cytokine, preventing it from interacting with its receptor in macrophages.

FIGURE 5.
FIGURE 5.

Decorin binds TGF-β and inhibits TGF-β binding to its receptor. a, Saturating curves of 125I-labeled TGF binding to macrophages cultured on a surface precoated with 10 μg/ml BSA or decorin. Binding experiments were performed as described in Materials and Methods. Each point was determined in triplicate and is presented as the mean ± SD. b, Scatchard analysis of 125I-labeled TGF-β in the presence of increasing concentrations of unlabeled TGF-β in macrophages cultured on a surface precoated with 10 μg/ml BSA or decorin. Each measurement was made in triplicate and is presented as the mean ± SD. The Scatchard analysis shows one representative of three independent experiments.

Next, we explored whether decorin could suppress the inhibitory capability of exogenous TGF-β to block macrophage activation. Treatment of macrophages with 10 ng/ml TGF-β was sufficient to completely block the expression of IFN-γ-induced MHC class II mRNA. However, when macrophages were cultured in the presence of decorin, TGF-β did not block MHC II expression (Fig. 6⇓a). Similar results were observed when we analyzed the effects of TGF-β and decorin on the expression of iNOS or IL-6 mRNA induced by LPS (Fig. 6⇓b).

FIGURE 6.
FIGURE 6.

Decorin blocks the immunosuppressive effect of TGF-β on macrophage activation. a, TGF-β suppresses IFN-γ-induced MHC II expression, and this effect is reversed by decorin. Macrophages were cultured on plates precoated with BSA or decorin. Once attached, cells were stimulated with 1 ng/ml TGF-β for 30 min and then activated with subsaturating amounts of IFN-γ for 6, 12, and 24 h. The expression of IA mRNAs was analyzed by Northern blot. b, TGF-β blocks LPS-induced iNOS and IL-6 expression, and this effect is inhibited by decorin. Macrophages were cultured in plates precoated with 10 μg/ml BSA or decorin. They were then stimulated with 1 ng/ml TGF-β for 30 min and activated with subsaturating amounts of LPS for the indicated times. The expression of iNOS and IL-6 mRNAs was analyzed by Northern blot. The results in a and b are representative of at least three independent experiments.

Our results show that decorin blocked the binding of TGF-β to the corresponding receptor, thereby reversing its inhibitory effects on macrophage activation. However, these results do not explain the increase in macrophage activation produced by decorin in the absence of exogenous TGF-β. However, an explanation was provided when IFN-γ- or LPS-activated macrophages were treated with blocking Abs against TGF-β. Like decorin, under these conditions anti-TGF-β Abs increased iNOS expression induced by subsaturating amounts of IFN-γ (Fig. 7⇓a) or LPS (Fig. 7⇓b). Anti-TGF-β and decorin had no effect on iNOS expression in the absence of macrophage activators.

FIGURE 7.
FIGURE 7.

The effects of decorin on macrophage activation are due to sequestration of endogenous TGF-β. Cells were cultured in plates precoated with 10 μg/ml BSA or decorin. After attachment to the plate, BSA-adhered cells were treated with 1 μg/ml anti-TGF-β polyclonal Abs for 30 min or were left untreated. Then BSA- and decorin-precoated plates were stimulated with subsaturating amounts of IFN-γ (a) or LPS (b) for 6 h or were left unstimulated, and the expression of iNOS was analyzed by Western blot analysis. Each experiment was performed twice. c, Expression of TGF-β after treatment with subsaturating amounts of IFN-γ (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-γ and LPS induced the expression of TGF-β in macrophages (Fig. 7⇑c). Since decorin did not modify this expression, the blocking effect of this proteoglycan was due to TGF-β sequestration and inhibition of TGF-β binding to its receptor.

Discussion

Under a range of external stimuli, macrophages proliferate, become activated and carry out their function, remain quiescent, or die through apoptosis. Like other immune cells, macrophages are produced in large excess, and only the few cells required to develop a functional activity survive. We found that IFN-γ not only activated macrophages, but also prevented the induction of apoptosis through the expression of the cdk inhibitor p21waf-1 and the arrest of cell cycling at the G1/S boundary (3).

In a previous study we observed that decorin, like IFN-γ, blocks macrophage proliferation and protects against apoptosis through the induction of p21waf1 (14). In the present study we found that decorin enhanced both IFN-γ- and LPS-mediated activation. This enhancing effect was originally linked to the capacity of decorin to increase cell adhesion on the basis that fibronectin, another ECM protein that induces cell adhesion, also inhibits macrophage proliferation. However, we observed that decorin increased the activation mediated by both LPS and IFN-γ, whereas fibronectin did not.

Because decorin-induced adhesion did not seem to be a sufficient mechanism to increase macrophage activation, we analyzed the repressive effect of this proteoglycan on TGF-β. This cytokine is produced by macrophages in an autocrine manner, and it down-regulates activation (26). TGF-β antagonizes IFN-γ-driven processes of macrophage activation, such as the production of H2O2, NO, the up-regulation of iNOS, the release of TNF-α, or the IFN-γ-induced death of intracellular microorganisms (27, 28, 29, 30). TGF-β also shows an inhibitory effect on IFN-γ-induced MHC class II genes and is mediated by a conserved proximal promoter element (31). The repressive effect of TGF-β on IFN-γ is based on cross-talk between the molecules involved in signal transduction pathways. The TGF-β/SMAD signaling cascades are inhibited by IFN-γ/STAT pathways and vice versa (32, 33). In addition, TGF-β inhibits LPS-induced activation of macrophages. This cytokine inhibits LPS-induced iNOS expression (34) or reduces the expression of proinflammatory cytokines during septic shock (35).

Our results also show that treatment of macrophages with decorin alone is sufficient to induce the mRNA expression of some cytokines, such as TNF-α and IL-1β, but it is not able to induce their secretion. This indicates that although treatment with decorin alone could modulate gene transcription, it does not regulate the post-transcriptional mechanisms involved in cytokine secretion. Increased expression and secretion of these inflammatory cytokines induced by decorin were only observed in the presence of macrophage activators such as LPS or IFN-γ.

Here we found that macrophages bound TGF-β with an affinity and number of binding sites per cell similar to those observed in other cell types (25). Decorin also blocked the binding of TGF-β to macrophages, which could be due to binding of decorin to TGF-β and inhibition of the interaction with the cell surface receptor. In vitro, decorin binds a variety of adhesive and nonadhesive proteins, including fibronectin, thrombospondin, various types of collagens, C1q, as well as TGF-β (8). Therefore, our results indicate that this proteoglycan inhibits the effects of TGF-β in macrophages. In response to subsaturating amounts of IFN-γ or LPS, decorin may compete with macrophages for the autocrine TGF-β produced. This could explain the beneficial effect of decorin on IFN-γ- or LPS-mediated activation.

Our findings further show that macrophages are subjected to negative regulation through the autocrine production of TGF-β. In fact, studies involving mouse models in which TGF-β was inactivated through disruption of the gene show an excessive inflammatory response (36), an increased expression of MHC II genes (37), and an increased production of NO (38). In addition, the inflammatory process in TGF-β1 knockout mice seems to be closely associated with the development of autoimmunity, as shown by the development of a massive mononuclear cell infiltration in multiple tissues, including lungs, heart, and salivary glands (36, 37, 38). All these data indicate that autocrine production of TGF-β plays a key role in the active suppression of inflammation in the absence of adequate proinflammatory stimuli. Therefore, macrophages in the absence of stimuli are in a preactivated state, which is maintained by inhibitory cytokines such as TGF-β.

Our results may have clinical and physiological relevance. Here we present a mechanism that blocks the endogenous inhibitor TGF-β. The presence of decorin in tissues could account for increased macrophage activation. These phagocytic cells play a critical role during inflammation. In the early stages, neutrophils are present at the inflammatory loci, but leave after 24–48 h. Later, macrophages reach these loci, where they remain until inflammation disappears (39), i.e., for as long as stimulated Th1 cells produce IFN-γ. In the late phases of inflammation, macrophages eliminate nonself structures, remove all debris (including apoptotic bodies), and remodel impaired tissues. However, during chronic inflammation, such as rheumatoid arthritis, macrophages play a key role in the pathogenesis (40). In these situations the persistence of these phagocytic cells may be related to the presence of molecules that block their deactivation. Several soluble mediators, such as TGF-β (28, 29, 30), IL-10 (30, 41), adenosine (19), etc., block macrophage activation. Macrophages are restrained from tissue-damaging activation by CD200R (a myeloid-specific receptor on the phagocytes) when it engages on other cells the glycoprotein CD200 (42, 43). Depending on the balance between activators and inhibitors, macrophages remain at the inflammatory loci and release enzymes or cytokines that could be deleterious for the articulation (44). In this context, decorin or other molecules of the ECM may contribute to the pathogenesis of chronic inflammation by blocking inhibitors. In this regard, in an animal model of experimental autoimmune encephalomyelitis, systemic administration of Abs specific for TGF-β identified a role for endogenous TGF-β in suppression of the disease (45).

Acknowledgments

We give special thanks to Dr. E. Ruoslahti (The Burnham Institute, La Jolla, CA) for the gift of the purified decorin used in this study. We also thank Dr. J. Gascon (Hospital Clinic, Barcelona, Spain) and Puleva Biotech (Granada, Spain) for their help with some reagents. We thank Tanya Yates for editorial help.

Footnotes

  • ↵1 This work was supported by grants from the Ministerio de Ciencia y Tecnología (BMC2001-3040 to A.C. and BMC2002-00737 to F.V.). M.C. was the recipient of a fellowship from the Fundació August Pi i Sunyer.

  • ↵2 M.C., M.C., and J.X. contributed equally to this work.

  • ↵3 Current address: Division of Genito-Urinary Oncology, M. D. Anderson Medical Center, Houston, TX 78957.

  • ↵4 Address correspondence and reprint requests to Dr. Antonio Celada, Research Institute of Biomedicine of Barcelona-Sciences Park, Josep Samitier 1-5, 08028 Barcelona, Spain. E-mail address: acelada{at}ub.edu

  • ↵5 Abbreviations used in this paper: ECM, extracellular matrix; iNOS, inducible NO synthase.

  • Received October 10, 2002.
  • Accepted February 24, 2003.

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