INFECTION AND IMMUNITY, Jan. 2011, p. 192–202 0019-9567/11/$12.00 doi:10.1128/IAI.00934-10 Copyright © 2011, American Society for Microbiology. All Rights Reserved.
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Granulocyte-Macrophage Colony-Stimulating Factor- and Tumor Necrosis Factor Alpha-Mediated Matrix Metalloproteinase Production by Human Osteoblasts and Monocytes after Infection with Brucella abortus䌤 Romina Scian,1 Paula Barrionuevo,1,2 Guillermo H. Giambartolomei,1,2 Carlos A. Fossati,1 Pablo C. Baldi,1* and M. Victoria Delpino1 Instituto de Estudios de la Inmunidad Humoral, Facultad de Farmacia y Bioquímica,1 and Laboratorio de Inmunogene´tica, Hospital de Clínicas Jose´ de San Martín, Facultad de Medicina,2 Universidad de Buenos Aires, Buenos Aires, Argentina Received 26 August 2010/Returned for modification 17 September 2010/Accepted 7 October 2010
Osteoarticular complications are common in human brucellosis, but the pathogenic mechanisms involved are largely unknown. Since matrix metalloproteinases (MMPs) are involved in joint and bone damage in inflammatory and infectious diseases, we investigated the production of MMPs by human osteoblasts and monocytes, either upon Brucella abortus infection or upon reciprocal stimulation with factors produced by each infected cell type. B. abortus infection of the normal human osteoblastic cell line hFOB 1.19 triggered a significant release of MMP-2, which was mediated in part by granulocyte-macrophage colony-stimulating factor (GM-CSF) acting on these same cells. Supernatants from infected osteoblasts exhibited increased levels of monocyte chemoattractant protein 1 and induced the migration of human monocytes (THP-1 cell line). Infection with B. abortus induced a high MMP-9 secretion in monocytes, which was also induced by heat-killed B. abortus and by the Omp19 lipoprotein from B. abortus. These effects were mediated by Toll-like receptor 2 and by the action of tumor necrosis factor alpha (TNF-␣) produced by these same cells. Supernatants from B. abortus-infected monocytes induced MMP-2 secretion in uninfected osteoblasts, and this effect was mediated by TNF-␣. Similarly, supernatants from infected osteoblasts induced MMP-9 secretion in uninfected monocytes. This effect was mediated by GM-CSF, which induced TNF-␣ production by monocytes, which in turn induced MMP-9 in these cells. These results suggest that MMPs could be potentially involved in the tissue damage observed in osteoarticular brucellosis. In different conditions, not only cytokines and chemokines but also matrix metalloproteinases (MMPs) are usually released within the inflammatory milieu. MMPs comprise a large family of Zn2⫹- and Ca2⫹-dependent endopeptidases whose capacity to degrade extracellular matrix is associated with tissue remodeling, chronic inflammation, tumor cell metastasis, and the progression of various infectious diseases (36, 37, 46). MMPs are secreted as inactive proenzymes and subsequently activated by proteolytic cleavage (7, 40). The transcription of several MMPs in most cells is induced by a wide range of growth factors and proinflammatory cytokines (15). While MMPs play an important role in facilitating the migration of innate inflammatory cells (12), excessive inflammation after infection may cause tissue damage due in part to increased levels of MMP activity (17). Of considerable importance in osteoarticular diseases are MMP-2 and MMP-9 (gelatinases A and B, respectively) that can degrade a variety of collagens including basement membrane (type IV collagen), denatured fibrillar type I collagen (gelatin) and type V collagen (42). Locally increased levels of MMPs have been found in several osteoarticular diseases, including rheumatic conditions (rheumatoid arthritis, osteoarthritis, and spondyloarthritis) and in infectious arthritis such as that observed in Lyme disease (5, 42, 50). Notably, in the synovial fluid of a patient with prepatellar bursitis due to Brucella we found a high gelatinase activity as revealed by zymography and the detection of high levels of MMP-9, which suggests that
Brucellosis is a multisystemic debilitating disease caused by Brucella spp., which may eventually become chronic. Humans usually get the infection from contact with infected animals and animal products, particularly milk and cheese (45). Brucellosis manifestations are mainly due to inflammatory phenomena, which may be found both in the acute and chronic phases of the disease and in virtually all of the affected organs (39). Osteoarticular involvement, including spondylitis, sacroiliitis, osteomyelitis, peripheral arthritis, bursitis, and tenosynovitis, represents the most common complication of brucellosis affecting up to 85% of patients (3, 17, 24, 29, 41, 48). While the clinical and imaging aspects of osteoarticular brucellosis have been described widely, the cellular and molecular pathogenic mechanisms of joint and bone disease caused by Brucella have been virtually ignored. A septic form of brucellar arthritis is supported by the isolation of Brucella spp. from synovial fluid or tissue from affected patients (20, 35). In addition, previous studies performed in our laboratory showed that Brucella spp. can infect and survive within human osteoblastic cell lines (11).
* Corresponding author. Mailing address: IDEHU, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956 4to. Piso, 1113 Buenos Aires, Argentina. Phone: 54-11-4964-8259. Fax: 54-11-4964-0024. E-mail:
[email protected]. 䌤 Published ahead of print on 18 October 2010. 192
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MMPs may be involved in the osteoarticular damage associated with Brucella infection (51). We have previously shown that Brucella can infect and survive within human osteoblasts and that this infection elicits the secretion of proinflammatory cytokines and chemokines (interlekin-8 [IL-8] and monocyte chemoattractant protein 1 [MCP-1], attractants for neutrophils and monocytes, respectively) (11). The study also revealed that cytokines produced by Brucella-infected monocytes induce chemokine secretion by osteoblasts, whereas factors produced by infected osteoblasts induce cytokines secretion by monocytes. The inflammatory trait of acute and chronic brucellosis (53), together with the detection of the bacterium in the inflamed tissues, suggests that Brucella stimulates a robust inflammatory response. Although Brucella lipopolysaccharide (LPS) has been found to be virtually devoid of proinflammatory activity (16), the production of proinflammatory cytokines by monocytes/macrophages, neutrophils, dendritic cells, astrocytes, and microglia is mainly induced by Brucella lipoproteins (4, 13, 14, 55, 56). In the present study we investigated the involvement of human osteoblasts and monocytes, and of cytokine networks between both cell types, in the induction of MMPs which may be relevant to the pathogenesis of osteoarticular brucellosis. We focused on the role of proinflammatory cytokines and bacterial components as mediators of bone damage through MMPs induction. MATERIALS AND METHODS Bacterial culture. Brucella abortus S2308 was grown as described previously (14). To obtain heat-killed B. abortus (HKBA), bacteria were washed five times for 10 min each in sterile phosphate-buffered saline, heat-killed at 70°C for 20 min, divided into aliquots, and stored at ⫺70°C until they were used. The total absence of B. abortus viability after heat killing was verified by the absence of bacterial growth on tryptose soy agar. All live Brucella manipulations were performed in biosafety level 3 facilities located at the Centro Nacional de Referencia del Sida, School of Medicine, University of Buenos Aires. Cell culture. The immortalized human fetal osteoblastic cell line hFOB 1.19 (abbreviated hFOB from now onwards) was obtained from the American Type Culture Collection (Rockville, MD). These cells have the ability to differentiate into mature osteoblasts expressing the normal osteoblast phenotype (24). The hFOB cell line was cultured as monolayers in a 5% CO2 atmosphere at 37°C in Dulbecco modified Eagle medium F-12 nutrient mixture (Gibco, Grand Island, NY) supplemented with 2 mM L-glutamine, 10% heat-inactivated fetal bovine serum (FBS; Gibco), 100 U of penicillin/ml, and 100 g of streptomycin/ml. The human monocytic cell line THP-1 was cultured in a 5% CO2 atmosphere at 37°C in RPMI 1640 (Gibco) supplemented as described above. All cell lines were seeded at 5 ⫻ 105 cells/well in 24-well plates. Lipoproteins and LPS. The lipidated and unlipidated forms of the 19-kDa outer membrane protein from B. abortus (L-Omp19 and U-Omp19, respectively) were obtained as described previously (14). Both recombinant proteins contained less than 0.25 endotoxin units per g of protein as assessed by the Limulus amebocyte lysate test (Associates of Cape Cod, East Falmouth, MA). The protein concentration was determined by the bicinchoninic acid method (Pierce, Rockford, IL) using bovine serum albumin (BSA) as standard. B. abortus S2308 LPS and Escherichia coli O111k58H2 LPS were provided by I. Moriyon (University of Navarra, Pamplona, Spain). The synthetic lipohexapeptide tripalmitoyl-S-glyceryl-Cys-Ser-Lys4-OH (Pam3Cys) was purchased from Boehringer Mannheim (Indianapolis, IN). Celular infection. hFOB cells were infected with B. abortus S2308 at different multiplicities of infection (MOIs; 100, 250, 500, and 1,000), and THP-1 cells were infected at an MOI of 100. After the bacterial suspension was dispensed, the plates were centrifuged for 10 min at 1,000 ⫻ g and then incubated for 2 h at 37°C under a 5% CO2 atmosphere. Cells were extensively washed with RPMI to remove extracellular bacteria and incubated in medium devoid of FBS and supplemented with BSA
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(0.01%), with 100 g of gentamicin/ml and 50 g of streptomycin/ml to kill extracellular bacteria (complete medium). Supernatants from infected cultures were harvested at 24 h postinfection (p.i.) to be used as conditioned medium and at 48 h p.i. for measuring MMPs. Zymography. Gelatinase activity was assayed by the method of Hibbs et al. (22). Briefly, 20 l of osteoblast or monocyte conditioned medium mixed with 5 l of sample buffer (0.25 M Tris [pH 6.8], 50% glycerol, 5% sodium dodecyl sulfate [SDS], and bromophenol blue crystals) were loaded per lane on 10% SDS–PAGE gels containing 1 mg of gelatin (Sigma-Aldrich, Argentina)/ml. After electrophoresis, the gels were washed with 50 mM Tris-HCl (pH 7.5)–2.5% Triton X-100 for 30 min and with 50 mM Tris-HCl (pH 7.5)–2.5% Triton X-100–5 mM CaCl2–1 M ZnCl2 for 30 min and then incubated with 50 mM Tris–HCl (pH 7.5)–2.5% Triton X-100–10 mM CaCl2–200 mM NaCl for 48 h at 37°C. This denaturation/renaturation step promotes MMP activity without proteolytic cleavage of pro-MMP-9. Gelatin activity was visualized by staining gels with 0.5% Coomassie blue. Unstained bands indicated the presence of gelatinase activity, and their position indicated the molecular weight of the enzymes involved. The identity of the candidate MMP was confirmed by a specific enzymelinked immunosorbent assay (ELISA) as explained below. Measurement of MMP-9 and MMP-2 levels. MMP-2 and MMP-9 present in conditioned media from hFOB and THP-1 cells, respectively, were quantified by sandwich ELISA using paired MMP-specific monoclonal antibodies according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN). Migration assay. Cell migration was evaluated by using 96-well microchemotaxis plates with 5-m-pore-diameter polycarbonate filters (Corning, Corning, NY). Monocytes (THP-1 cells, 106 cells/ml) were placed in the upper well of the chambers, and the indicated stimuli (dilutions of culture supernatants from B. abortus-infected hFOB) were placed in the lower wells. Migration was scored by counting the number of monocytes that had reached the bottom well after 2 h. Migration toward N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP, 10⫺7 M) (Sigma-Aldrich) served as a positive control. The number of migrating cells was expressed as: chemoattractant index ⫽ number of migrating cells to the tested medium/number of migrating cells to fresh culture medium. Stimulation with conditioned medium. Culture supernatants from Brucellainfected THP-1 monocytes (MOI ⫽ 100) and from Brucella-infected osteoblastic hFOB cells (MOI ⫽ 1,000) were harvested at 24 h p.i., sterilized by filtration through a 0.22-m-pore-size nitrocellulose filter, and used to stimulate noninfected hFOB and THP-1 cells, respectively. Supernatants were used diluted 1/2, 1/5, 1/10, or 1/100 in complete medium. After 48 h, the supernatants from these stimulated cultures were harvested to measure MMPs. Measurement of cytokine concentrations. Human interleukin-1 (IL-1), IL-6, IL-8, monocyte chemotactic protein 1 (MCP-1), tumor necrosis factor alpha (TNF-␣), and granulocyte-macrophage colony-stimulating factor (GMCSF) were measured in culture supernatants by sandwich ELISA, using paired cytokine-specific monoclonal antibodies, according to the manufacturer’s instructions (BD Pharmingen, San Diego, CA). Blocking of Toll-like receptors (TLRs). THP-1 cells (106/ml) were incubated with 20 g of anti-human TLR2 antibody (clone TL2.1), anti-human TLR4 (clone HTA125), or an IgG2a isotype control (eBioscience)/ml for 30 min at 37°C and then incubated with E. coli LPS (100 ng/ml), Pam3Cys (50 ng/ml), HKBA (108 bacteria/ml), or L-Omp19 (1,000 ng/ml) in a final volume of 0.4 ml. Untreated cells were also incubated in parallel with U-Omp19 (1,000 ng/ml) and B. abortus LPS (1,000 ng/ml) as controls. Cultures were incubated for 24 h and supernatants were assayed for cytokine production as described. Blocking of cytokines and their receptors. Neutralization experiments were performed with an antibody against the human TNF receptor I (anti-TNFRI, clone 16805) or its isotype control (both from R&D Systems), an anti-TNF-␣ neutralizing antibody (clone MAb1) or its isotype control (both from BD Biosciences, San Diego, CA), an anti-GM-CSF neutralizing antibody (clone BVD223B6) or its isotype control (BD Biosciences), or the natural antagonist IL-1Ra (R&D Systems). In neutralization experiments with anti-TNFRI or IL-1Ra, cells were preincubated with these reagents for 1 h at 37°C before stimulation with conditioned media or with Brucella antigens, and in those using neutralizing anti-TNF-␣ antibody or anti-GM-CSF antibody, the conditioned medium was preincubated with the corresponding antibody (or isotype control) for 1 h at 37°C before use. Gelatinase activity under native conditions. Gelatinase activity in unprocessed culture supernatants (native conditions) was measured by using a gelatinase/ collagenase fluorometric assay kit (EnzChek; Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The EnzChek kit contains DQ gelatin, a fluorescein-conjugated gelatin so heavily labeled with fluorescein that fluorescence is quenched. When this substrate is digested by gelatinases or collagenases it yields highly fluorescent peptides, and fluorescence increase is proportional to
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proteolytic activity. Collagenase purified from Clostridium histolyticum provided in the assay kit serves as a control enzyme. Plates were read in a fluorescence plate reader (Victor3; Perkin-Elmer, Waltham, MA). Statistical analysis. Statistical analysis was performed with one-way analysis of variance, followed by a post hoc Tukey test using GraphPad Prism 4.0 software. The data are represented as means ⫾ the standard deviations (SD).
RESULTS Brucella abortus induces a GM-CSF-dependent release of MMP-2 in osteoblasts. Infections of the hFOB cell line with B. abortus S2308 were performed at MOIs of 100, 250, 500, and 1,000. The ability of B. abortus to infect the hFOB cell line was similar to that previously found for MG-63 and SaOS-2 osteosarcoma cell lines (11), and the magnitude of the infection (intracellular CFU) was directly related to the MOI used (Fig. 1A). Significantly increased levels of MMP-2 were detected at 48 h p.i. in the supernatants of infected cells (Fig. 1B and C). The magnitude of MMP-2 release was directly related to the MOI used. Proinflammatory cytokines and growth factors have been shown to stimulate MMPs secretion by different cellular types (20, 25, 26, 43), in some instances in an autocrine fashion (2, 21). To test whether MMP-2 induction in Brucella-infected osteoblasts may be mediated by cytokines or growth factors produced by these same cells, the levels of IL-1, IL-6, TNF-␣, and GM-CSF were measured in culture supernatants. In agreement with our previous findings in osteosarcoma cell lines (11) we found that hFOB cells secrete GM-CSF in response to Brucella infection in a MOI-dependent fashion (Fig. 1D). In contrast, IL-1, IL-6, and TNF-␣ were not detected in culture supernatants from Brucella-infected hFOB cells (not shown). To test whether GM-CSF secreted by Brucella-infected osteoblasts acts on these same cells to induce MMP-2 production, hFOB cells were infected in the presence of either an anti-GM-CSF monoclonal neutralizing antibody or an isotype control. As shown in Fig. 1E, neutralization of GM-CSF significantly reduced the ability of Brucella infection to stimulate MMP-2 production by osteoblasts, whereas the isotype control had no effect. Supernatants from B. abortus-infected osteoblasts induce monocyte migration. In agreement with previous studies using osteosarcoma cell lines (11), we found that hFOB cells secrete MCP-1 in response to Brucella infection (Fig. 2A). Therefore, additional experiments were conducted to evaluate whether supernatants from infected osteoblasts were able to induce monocyte migration. For this purpose, monocytic THP-1 cells were placed in the top wells of a microchemotaxis plate, and different dilutions of supernatants from infected or noninfected osteoblasts were placed in the bottom wells of the chamber. Migration was scored by counting the number of cells that reached the lower chamber after a 2-h incubation period. In wells containing supernatants from infected osteoblasts and in the fMLP control the chemoattractant index was significantly higher (P ⬍ 0.01) than the background migration, while migration in chambers with supernatants from noninfected osteoblasts remained at background levels (Fig. 2B). Therefore, B. abortus-infected osteoblasts produce chemoattractants, including MCP-1, that may induce monocyte migration to the site of infection.
FIG. 1. (A) Infection and replication of B. abortus within human osteoblasts (hFOB cell line). After infection at different MOIs, cells were incubated with antibiotics to kill extracellular bacteria. Cell lysates obtained at different times postinfection (p.i.) were plated on agar to determine intracellular CFU. (B and C) MMP-2 production at 48 h p.i. by B. abortus-infected osteoblasts as determined by ELISA (B) and zymography (C). (D) GM-CSF production by B. abortusinfected osteoblasts at 48 h p.i. (E) Inhibition of MMP-2 secretion by B. abortus-infected osteoblasts in the presence of an anti-GM-CSF neutralizing antibody or an isotype control. The data shown are from a representative experiment of five performed. *, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001 versus control.
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FIG. 2. (A) MCP-1 production at 48 h p.i. by human osteoblasts infected with B. abortus at different MOIs. Migration of monocytes induced by different amounts (pure, 1/2 and 1/5 dilutions) of culture supernatants from Brucella-infected or uninfected osteoblasts, as measured in a microchemotaxis plate. Migrated cells were counted at 2 h, and results are expressed as the chemoattractant index (number of cells that migrated to conditioned medium divided by the number of cells that migrated to culture medium). (B) Migration toward N-formyl-L-methionyl-L-leucylphenylalanine (fMLP) served as a positive control. (C and D) MMP-9 production at 48 h p.i. by B. abortus-infected monocytes was determined by ELISA (C) and zymography (D). The data shown are from a representative experiment of five performed. *, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001 versus control.
B. abortus induces the release of MMP-9 by monocytes. In view of the ability of Brucella-infected osteoblasts to produce monocyte-attracting factors that could potentially recruit monocytes to the site of infection, we decided to test whether B. abortus infection induces MMP activity in monocytes. As shown in Fig. 2C and D, infection with B. abortus induced MMP-9 secretion in the human monocytic THP-1 cell line. MMP-9 was secreted at high levels and in an MOI-dependent fashion. Structural components of Brucella induce MMP-9 production by monocytes but not MMP-2 by osteoblasts. Next we assessed whether MMP-2 production by osteoblasts and MMP-9 production by monocytes requires bacterial viability or, alternatively, can be induced by structural components of Brucella spp. To accomplish the later goal, we examined the role of specific bacterial components, and heat-killed B. abortus S2308 (HKBA). Osteoblasts stimulated with HKBA did not produce detectable levels of MMP-2 (not shown), whereas elevated levels of MMP-9 expression were detected in supernatants of monocytes at 48 h after exposure to different doses of HKBA (Fig. 3). Since proinflammatory cytokines can induce MMPs production in monocytes (20, 52) and Brucella lipoproteins (and not LPS) are responsible for the proinflammatory cytokine induction in macrophages and dendritic cells (14, 56), we hypothesized that lipoproteins could be the constitutive components of Brucella involved in MMP-9 induction in THP-1 cells. To test this hypothesis, we used recombinant L-Omp19 as a Brucella lipoprotein model. Monocytes were incubated with L-Omp19, and culture supernatants were harvested 48 h later to measure MMP-9 expression by zymography and ELISA. As shown in
Fig. 3, L-Omp19 induced MMP-9 expression in a dose-dependent fashion. Significant production was seen with as little as 10 ng of L-Omp19/ml, and maximum production was observed with 1,000 ng/ml. MMP-9 induction was dependent on the lipidation of L-Omp19, since U-Omp19 failed to upregulate MMP-9 expression even at a concentration of 1,000 ng/ml (Fig. 3). To ascertain whether the effects elicited by L-Omp19 could be extended to all B. abortus lipoproteins, MMP-9 production was also evaluated in monocytes incubated with a synthetic lipohexapeptide (Pam3Cys) that mimics the structure of the lipoprotein lipid moiety. As shown in Fig. 3, Pam3Cys also stimulated MMP-9 expression by monocytes. These results indicate that the Pam3-modified cysteine is the molecular structure of lipoproteins that induces MMP-9 upregulation. On the contrary, B. abortus LPS did not induce MMP-9 production even when used at high doses (1,000 ng/ml), whereas LPS from E. coli did (Fig. 3). MMP-9 induction by L-Omp19 is mediated by TLR2. Previous studies have shown that TLR2 mediates responses to HKBA and B. abortus lipoproteins in cells of the human monocytic lineage (14, 56). Consequently, we analyzed the role of TLR2 in the MMP-9 production induced by HKBA and LOmp19 in THP-1 cells. Monocytes were preincubated with anti-TLR2 or anti-TLR4 neutralizing antibodies or the corresponding isotype controls and were then cultured with HKBA or L-Omp19. The production of MMP-9 was evaluated in the culture supernatants by zymography and ELISA. E. coli LPS and Pam3Cys were used as control agonists. As expected, preincubation of THP-1 cells with anti-TLR4 significantly blocked (P ⬍ 0.01) the E. coli LPS-mediated production of MMP-9, whereas anti-TLR2 inhibited significantly (P ⬍ 0.01) the MMP
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FIG. 3. MMP-9 production by human monocytes stimulated with HKBA (106 to 109 bacteria/ml), E. coli LPS (100 ng/ml), B. abortus LPS (1,000 ng/ml), L-Omp19 (10, 100, or 1,000 ng/ml), U-Omp19 (1,000 ng/ml), Pam3Cys (50 ng/ml), or nonstimulated (Control), as determined by ELISA (A) and zymography (B). The data shown are from a representative experiment of five performed. **, P ⬍ 0.01; ***, P ⬍ 0.001 versus control.
production induced by Pam3Cys (Fig. 4). Preincubation of THP-1 cells with anti-TLR2 significantly blocked the L-Omp19-mediated production of MMP-9 and also the HKBA-mediated production of this enzyme. In contrast, preincubation with the anti-TLR4 antibody did not modify significantly the inducing effect of L-Omp-19 or HKBA on MMP-9 production. Isotype control antibodies had no effect on any of the responses investigated. These results indicated that, in THP-1 cells, the secretion of MMP-9 induced by B. abortus depends on lipoprotein recognition by TLR2. L-Omp19 induces MMP-9 production through TNF-␣ induction. In agreement with previous reports (14), significantly elevated levels of TNF-␣, IL-1, and IL-6 expression were detected in culture supernatants of THP-1 cells at 24 h after exposure to either live B. abortus S2308 (MOI of 25 to 100) or to HKBA (not shown). Since TNF-␣ and IL-1 are known to induce the production of MMP-9 by different cell types, including macrophages (28, 45, 54), we decided to determine the contributions of these cytokines to the induction of MMP-9 by Brucella antigens in monocytes. To address this issue, we evaluated the effects of
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FIG. 4. TLR2-dependency of MMP-9 expression induced by HKBA and L-Omp19. THP-1 cells were incubated with anti-TLR2 or anti-TLR4 blocking antibodies or an isotype control for 30 min before the addition of HKBA (107 bacteria/ml), E. coli LPS (EcLPS) (100 ng/ml), L-Omp19 (100 ng/ml), or Pam3Cys (50 ng/ml). After 48 h of culture, MMP-9 expression was assessed in supernatants by ELISA (A) and zymography (B). The data shown are from a representative experiment of five performed. ***, P ⬍ 0.001 versus anti-TLR2 or anti-TLR4 treatment (whichever is applicable).
blocking the action of these cytokines by using a TNF-␣ receptor I (TNFRI) blocking antibody or its isotype control and the natural antagonist IL-1Ra (to block the IL-1 receptor). As shown in Fig. 5, blocking of TNFRI significantly reduced the ability of HKBA, L-Omp-19 and Pam3Cys to induce MMP-9 production in monocytes, whereas the isotype control had no effect. On the other hand, preincubation with the natural antagonist IL-1Ra did not affect the MMP-9 secretion induced by these stimulants (not shown). Supernatants from B. abortus-infected monocytes induce MMP-2 production by osteoblasts through TNF-␣. Since Brucella-infected osteoblasts may attract monocytes to the site of infection (as suggested by results shown in Fig. 2A and B), we decided to study the effects of cytokines secreted by monocytes on the production of gelatinases by osteoblasts, and vice versa. Therefore, media from B. abortus-infected osteoblasts and monocytes were harvested at 24 h p.i, sterilized by filtration, and used to stimulate monocytes and osteoblasts, respectively. The addition of supernatants from B. abortus-infected monocytes at different proportions (1/2 to 1/10) to uninfected osteoblasts induced a significant secretion of MMP-2 by the later cells compared to that in unstimulated cultures. In contrast, MMP-2 secretion was not induced when osteoblasts were
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FIG. 5. MMP-9 expression induced by HKBA and L-Omp19 is TNF-␣ dependent. THP-1 cells were incubated with a blocking antibody against the TNF-␣ receptor I (TNFRI) or its isotype control for 30 min before the addition of HKBA (107 bacteria/ml), E. coli LPS (EcLPS) (100 ng/ml), L-Omp19 (100 ng/ml), or Pam3Cys (50 ng/ml). After 48 h of culture, MMP-9 expression was assessed in supernatants by ELISA (A) and zymography (B). The data shown are from a representative experiment of five performed. **, P ⬍ 0.01; ***, P ⬍ 0.001 versus anti–TNFR1 treatment.
stimulated with supernatants from noninfected monocytes (Fig. 6A and B). Since TNF-␣ and IL-1 are known to induce MMP production by different cell types, the levels of these cytokines were measured by ELISA in supernatants of Brucella-infected THP-1 cells. Both cytokines were detected in supernatants from THP-1 cells infected at an MOI of 100 (4,500 ⫾ 240 pg/ml for TNF-␣; 72 ⫾ 6 pg/ml for IL-1). To confirm that such TNF-␣ levels can stimulate MMP-2 secretion by osteoblasts, these cells were incubated in the presence of supernatants from infected monocytes preincubated or not for 1 h with either an anti-TNF-␣ monoclonal neutralizing antibody or an isotype control. As shown in Fig. 6C neutralization of TNF-␣ reduced the ability of supernatants to stimulate MMP-2 secretion by osteoblasts, whereas the isotype control had no effect. In contrast, preincubation with the natural antagonist IL-1Ra did not affect the MMP-2 secretion induced by these supernatants (not shown). Supernatants from B. abortus-infected osteoblasts induce MMP-9 production by monocytes due largely to GM-CSF. The inverse experiment, the stimulation of noninfected monocytes with supernatants from infected osteoblasts, was also per-
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FIG. 6. MMP-2 production by osteoblasts stimulated with supernatants from B. abortus-infected monocytes (added at a 1/2, 1/5, or 1/10 proportion) or from uninfected monocytes was determined by ELISA (A) and zymography (B). Inhibition of the stimulating effect by pretreatment of supernatants with an anti-TNF-␣ neutralizing antibody or an isotype control for 1 h before addition to osteoblasts (C). **, P ⬍ 0.01; ***, P ⬍ 0.001 versus control (unstimulated cells).
formed. Supernatants added at different proportions induced the production of MMP-9 by THP-1 cells compared to unstimulated cultures (Fig. 7A). In contrast, MMP-9 secretion was not stimulated by supernatants from noninfected osteoblasts. GM-CSF has been shown to stimulate MMP-9 production by monocytes (25, 54). In addition, we have shown here (Fig. 1D) and in a previous study (11) that GM-CSF is secreted by osteoblastic cell lines in response to Brucella infection. To evaluate whether GM-CSF may be involved in the ability of conditioned media from Brucella-infected hFOB osteoblasts to stimulate MMP-9 in monocytes, these conditioned media were preincubated or not with an anti-GM-CSF monoclonal neutralizing antibody (or an isotype control) before being used to stimulate monocytes. As shown in Fig. 7B and C, neutralization of GM-CSF significantly reduced the ability of hFOB supernatants to stimulate MMP-9 secretion by monocytes, whereas the isotype control had no effect. Supernatants from B. abortus-infected osteoblasts induce MMP-9 production by monocytes indirectly through TNF-␣ induction. Since GM-CSF has been shown to stimulate TNF-␣
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FIG. 7. MMP-9 production by monocytes stimulated with supernatants from B. abortus-infected osteoblasts (added at a 1/2, 1/5, or 1/10 proportion) or from uninfected osteoblasts, as determined by zymography (A). Inhibition of the stimulating effect by pretreatment of supernatants with a neutralizing antibody to GM-CSF or an isotype control for 1 h before addition to monocytes determined by ELISA (B) and zymography (C). *, P ⬍ 0.05 versus untreated supernatants.
and IL-1 secretion by monocytes (6, 10, 11), and these cytokines have been shown to stimulate MMP-9 secretion by monocytes (27, 35), we evaluated whether the stimulating effect of osteoblast-derived GM-CSF on MMP-9 production by monocytes is mediated by the induction of TNF-␣ and/or IL-1 in the later cells. In agreement with our previous findings with the osteosarcoma cell lines (11), we found that stimulation of monocytes with supernatants from Brucella-infected hFOB osteoblasts induced TNF-␣ and IL-1 production by THP-1 cells compared to unstimulated cultures (Fig. 8A and B) and that this induction depended on GM-CSF (Fig. 8C and D). In contrast, such induction was not produced by supernatants of uninfected hFOB cells. In addition, neither TNF-␣ nor IL-1 were detected in supernatants from infected or uninfected osteoblasts. To determine whether TNF-␣ or IL-1 produced by the
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stimulated monocytes mediate the inducing effect of GM-CSF on MMP-9 production by these cells, stimulation experiments were performed in which the receptors of these cytokines were blocked by preincubation of monocytes with an anti-TNFRI antibody or the natural antagonist IL-1Ra, respectively. As shown in Fig. 9, blocking of TNF-␣ receptor significantly reduced the ability of supernatants from Brucella-infected osteoblasts to induce MMP-9 secretion by monocytes. In contrast, blocking of IL-1 receptor with IL-1Ra had no effect (data not shown). Overall, these results confirmed that GM-CSF produced by Brucella-infected osteoblasts induces TNF-␣ production in monocytes, which acts on the later cells to induce MMP-9. Supernatants from Brucella-infected osteoblasts and monocytes cleave gelatin in the fluid phase. In vivo the activity of MMPs is counterbalanced by the activity of tissue inhibitors including TIMPs (7). Therefore, the net gelatinase or collagenase activity in a complex sample, such as culture supernatants, depends on the balance between MMP and TIMP activities. This net activity is not revealed by zymography, since MMPTIMP complexes may dissociate during gel electrophoresis. To assess whether the environment surrounding Brucella-infected monocytes or osteoblasts has an increased net gelatinase activity, culture supernatants from these cells were incubated with a nonfluorescent gelatin-fluorescein conjugate, and the fluorescence unmasked as a consequence of gelatin degradation was measured in a fluorometer. Enzymatic activity (measured as fluorescence) increased significantly in supernatants of Brucella-infected THP-1 monocytes or hFOB osteoblasts compared to uninfected cells, and the increase was somewhat MOI dependent. In the case of osteoblasts, whereas basal activity in uninfected cells was 2,350 ⫾ 12 fluorescence units, it reached 11,207 ⫾ 73, 11,688 ⫾ 170, 12,452 ⫾ 118, and 12,585 ⫾ 72 U for MOIs 100, 250, 500, and 1,000, respectively (all P ⬍ 0.001 versus uninfected control). In the case of monocytes, while basal activity was 2,602 ⫾ 45 fluorescence units, it reached 10,021 ⫾ 74, 11,977 ⫾ 120, 12,592 ⫾ 40, and 13,601 ⫾ 61 U for MOIs 10, 25, 50, and 100, respectively (all P ⬍ 0.001 versus uninfected control). These results showed that Brucella infection leads to a net increase of gelatinase activity in the vicinity of infected monocytes and osteoblasts. DISCUSSION In most cases of septic arthritis and osteomyelitis, bone and joint damage results from the inflammatory reaction elicited by the infection, including an increased MMP activity. Locally increased levels of MMPs have been found in arthritis associated with Lyme disease (5) and in periodontitis due to different bacteria (47). In these pathological processes, the sources of MMPs may be resident cells, such as osteoblasts, osteoclasts, synoviocytes, and chondrocytes, as well as phagocytes attracted to the inflammatory focus. Regarding phagocyte attraction, it is interesting that infected osteoblasts have been shown to produce proinflammatory chemokines (11, 31). The potential contribution of MMPs to tissue damage in osteoarticular brucellosis has not been assessed. Osteoblasts have been shown to produce several MMPs, among which MMP-2 is particularly important because it degrades type I collagen present in bone
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FIG. 8. GM-CSF produced by B. abortus-infected osteoblasts induces TNF-␣ and IL-1 production in monocytes. TNF-␣ (A) and IL-1 (B) production by monocytes stimulated with supernatants from B. abortus-infected osteoblasts. This stimulating effect is inhibited by pretreatment of supernatants with a neutralizing antibody to GM-CSF for 1 h before addition to monocytes (C and D, respectively).
and type II collagen present in cartilage (9). In the present study an increased MMP-2 activity was detected in supernatants from B. abortus-infected osteoblasts. Previous studies have shown that in several cell lines MMP production can be stimulated by GM-CSF (25, 26). Since this factor is produced by Brucella-infected osteoblasts, as shown here and also in our previous study (11), we assessed whether osteoblast-derived GM-CSF may induce MMP-2 in these same cells. Neutralization assays with specific antibodies demonstrated that GMCSF is a major mediator of MMP-2 production by osteoblasts upon Brucella infection. While other cytokines such as TNF-␣ and IL-1 are known to induce MMP-2 secretion by osteoblasts (33, 44), we only investigated the role of GM-CSF since Brucella-infected osteoblasts do not produce detectable levels of TNF-␣ or IL-1 as shown here and in our previous study (11). As mentioned above, phagocytes attracted to the site of infection also produce tissue-degrading MMPs. In agreement with our previous studies with the human osteoblastic cell lines SaOS-2 and MG-63 (11), in the present study we found that normal human osteoblasts (hFOB 1.19 cell line) respond to B. abortus infection with significant production of MCP-1, a chemoattractant for monocytes. Moreover, we
found that conditioned media from Brucella-infected osteoblasts induce monocyte/macrophage migration in vitro. Notably, a nonspecific inflammatory infiltrate has been observed in synovial membrane and bone in patients with brucellar arthritis and osteomyelitis, respectively (29). Therefore, Brucella-infected osteoblasts may be involved in matrix degradation not only by their MMP-2 production but also by their ability to recruit monocytes to the infectious focus. Recruited monocytes may mediate tissue damage through the production of MMP-9 involved in bone resorption and also through the secretion of proinflammatory cytokines that can induce MMPs in other cell types (see below). In the present study we found that human monocytes (THP-1 cell line) respond to Brucella infection with the production of MMP-9, which is known to degrade type V collagen present in bone (8, 32, 52). Such response was also elicited by HKBA, clearly indicating the involvement of one or more structural components of Brucella. Since proinflammatory cytokines are known to induce MMPs production in monocytes (20, 52) and since Brucella lipoproteins induce the production of proinflammatory cytokines in several cell types (13, 14, 56), we hypothesized that such lipoproteins
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FIG. 9. The induction of MMP-9 in monocytes by supernatants from infected osteoblasts is mediated through TNF-␣ induction. Monocytes were incubated with a blocking antibody against TNF-␣ receptor 1 (TNFR1) or with an isotype control before addition of supernatants from B. abortus-infected osteoblasts. Levels of MMP-9 were determined by ELISA (A) or zymography (B) in the supernatants of stimulated monocytes. **, P ⬍ 0.01; ***, P ⬍ 0.001 versus monocytes not pretreated with anti-TNFR1 (untreated).
could also mediate MMP-9 induction in THP-1 cells. We found that L-Omp19, but not its unlipidated form, induced MMP-9 production by this monocytic cell line. The same effect was observed for Pam3Cys, a synthetic lipohexapeptide that mimics the core structure of bacterial lipoproteins. In addition, neutralization experiments with an anti-TNFRI antibody or IL-1Ra clearly showed that the induction of MMP-9 in monocytes treated with HKBA or L-Omp19 was mediated by TNF-␣ but not by IL-1. Previous studies have shown that the cytokine production induced by HKBA and lipoproteins in cells of the monocytic linage depends on TLR2 stimulation (14). In line with these previous findings, in the present study the production of MMP-9 by monocytes depended on TLR-2 stimulation. Overall, these results showed that TNF-␣ produced by human monocytes in response to Brucella lipoproteins can induce these cells to express MMP-9. Since the B. abortus genome contains at least 80 genes encoding putative lipoproteins (19), many of which are expressed in the bacterial outer membrane, the concentration of Brucella lipoproteins in the infectious focus within the bone may be sufficient to exert the biological effects described above. In vivo, the activity of MMPs is counterbalanced by the activity of tissue inhibitors, including TIMPs (7). In osteoarticular infections or inflammatory conditions, TIMPs usually do not increase to the same extent as MMPs do, thus
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resulting in an increased MMP/TIMP ratio, which aggravates cartilage breakdown and promotes joint destruction (23, 30). In line with those findings, supernatants from Brucella-infected monocytes or osteoblasts produced gelatin breakdown when assayed in fluid phase under native conditions, in which MMP-TIMP complexes are not dissociated, as occurs during gel electrophoresis. As mentioned above, monocytes may be attracted to the site of infection by MCP-1 produced by Brucella-infected osteoblasts. In the in vivo situation, attracted and resident monocytes may respond to Brucella antigens with the production of proinflammatory cytokines. It has been shown that TNF-␣ and/or IL-1 induce the expression of several MMPs in different cell types, including osteoblasts, chondrocytes, synovial fibroblasts, monocytes, and many other cells (1, 20, 34, 38, 49, 52). Our results showed that supernatants from Brucella-infected monocytes stimulate osteoblasts to secrete MMP-2 and that TNF-␣ is a major mediator of this stimulating effect. In agreement with previous reports, IL-1 was not involved in MMP-2 induction in osteoblasts (18, 44). We hypothesized that, in addition, factors produced by infected osteoblasts could potentially influence MMP production by monocytes. In fact, we found that culture supernatants from Brucella-infected osteoblasts induced MMP-9 production by monocytes. Since such supernatants contained GM-CSF, which has been implicated in MMP induction in monocytes (25, 54), we tested whether GM-CSF was involved in MMP-9 induction. Neutralization experiments demonstrated that this induction was largely due to GMCSF present in osteoblasts conditioned medium. Since our previous studies had shown that GM-CSF also mediates the induction of TNF-␣ and IL-1 in THP-1 cells upon stimulation with conditioned medium from Brucella-infected osteoblasts (11), and taking into account that TNF-␣ and IL-1  can induce MMP-9 production in monocytes (27, 35), we evaluated whether the stimulating effect of osteoblast-derived GM-CSF on MMP-9 production by monocytes was mediated by these cytokines. Our results showed that the effect of GM-CSF was indirect, since this factor induced TNF-␣ secretion by monocytes, which in turn stimulated MMP-9 secretion in these cells. In conclusion, the present study clearly demonstrated that B. abortus infection induces MMP-2 secretion by osteoblasts and MMP-9 secretion by monocytes. Although B. abortus lipoproteins are responsible for MMP-9 induction by stimulating monocytes to secrete TNF-␣, which in turn induces MMP-9 production in these cells, MMP-2 secretion by osteoblasts depended on bacterial viability and was largely mediated by GM-CSF produced by the infected osteoblasts. We have also shown that TNF-␣ produced by Brucella-infected or lipoprotein-stimulated monocytes can also induce MMP-2 secretion by osteoblasts. In the inverse case, Brucella-infected osteoblasts produce GM-CSF which stimulates monocytes to produce TNF-␣, which in turn induces MMP-9 production in the later cells. Based on these findings, we propose a model for the production of MMPs by osteoblasts and monocytes during Brucella infection (Fig. 10). Based on the results obtained in the present study, we hypothesize that induction of MMP-2 and MMP-9 upon
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FIG. 10. Proposed model for the production of MMPs by osteoblasts and monocytes during Brucella infection. Osteoblasts would produce MMP-2 not only in response to infection but also in response to TNF-␣ produced by adjacent infected monocytes. MMP-2 production by infected osteoblasts would depend on the autocrine action of GM-CSF. Monocytes would produce MMP-9 in response to infection and also in response to GM-CSF produced by adjacent infected osteoblasts. The later effect would be mediated by the autocrine action of TNF-␣ produced by monocytes in response to GM-CSF.
Brucella infection could potentially contribute to the bone and joint destruction observed in patients with osteoarticular complications of brucellosis. ACKNOWLEDGMENTS We thank Horacio Salomo ´n and the staff of the Centro Nacional de Referencia del Sida, University of Buenos Aires, for their assistance with biosafety level 3 laboratory use. This study was supported by grants PICT2006-0517, PICT200538237, PICT2007-0139 and PICT2006-1335 from the Agencia Nacional of Promocio ´n Científica y Tecnolo ´gica (ANPCYT; Argentina) and by PIP112-200801-02706 from the Consejo Nacional de Investigacio ´n Científica y Tecnolo ´gica (CONICET). R.S. is recipient of a fellowship from Agencia Nacional of Promocio ´ n Científica y Tecnolo ´ gica (ANPCYT). M.V.D., P.B., G.H.G., C.A.F., and P.C.B. are members of the Research Career of CONICET. REFERENCES 1. Alsalameh, S., R. J. Amin, E. Kunisch, H. E. Jasin, and R. W. Kinne. 2003. Preferential induction of prodestructive matrix metalloproteinase-1 and proinflammatory interleukin 6 and prostaglandin E2 in rheumatoid arthritis synovial fibroblasts via tumor necrosis factor receptor-55. J. Rheumatol. 30:1680–1690. 2. Arechavaleta-Velasco, F., D. Ogando, S. Parry, and F. Vadillo-Ortega. 2002. Production of matrix metalloproteinase-9 in lipopolysaccharide-stimulated human amnion occurs through an autocrine and paracrine proinflammatory cytokine-dependent system. Biol. Reprod. 67:1952–1958. 3. Aydin, M., A. Fuat Yapar, L. Savas, M. Reyhan, A. Pourbagher, T. Y. Turunc, Y. Ziya Demiroglu, N. A. Yologlu, and A. Aktas. 2005. Scintigraphic findings in osteoarticular brucellosis. Nucleic Med. Commun. 26:639–647. 4. Barrionuevo, P., J. Cassataro, M. V. Delpino, A. Zwerdling, K. A. Pasquevich, C. Garcia Samartino, J. C. Wallach, C. A. Fossati, and G. H. Giambartolomei. 2008. Brucella abortus inhibits major histocompatibility complex class II expression and antigen processing through interleukin-6 secretion via Toll-like receptor 2. Infect. Immun. 76:250–262. 5. Behera, A. K., E. Hildebrand, J. Scagliotti, A. C. Steere, and L. T. Hu. 2005. Induction of host matrix metalloproteinases by Borrelia burgdorferi differs in human and murine Lyme arthritis. Infect. Immun. 73:126–134. 6. Bost, K. L., J. L. Bento, J. K. Ellington, I. Marriott, and M. C. Hudson. 2000. Induction of colony-stimulating factor expression following Staphylococcus or Salmonella interaction with mouse or human osteoblasts. Infect. Immun. 68:5075–5083. 7. Brinckerhoff, C. E., and L. M. Matrisian. 2002. Matrix metalloproteinases: a tail of a frog that became a prince. Nat. Rev. Mol. Cell. Biol. 3:207–214. 8. Burrage, P. S., and C. E. Brinckerhoff. 2007. Molecular targets in osteoarthritis: metalloproteinases and their inhibitors. Curr. Drug Targets 8:293– 303. 9. Burrage, P. S., K. S. Mix, and C. E. Brinckerhoff. 2006. Matrix metalloproteinases: role in arthritis. Front. Biosci. 11:529–543.
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