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The Journal of Immunology, 2003, 171: 5579-5586.
Copyright © 2003 by The American Association of Immunologists

Unopposed Matrix Metalloproteinase-9 Expression in Human Tuberculous Granuloma and the Role of TNF-{alpha}-Dependent Monocyte Networks1

Nicholas M. Price*, Robert H. Gilman*,{dagger},{ddagger}, Jasim Uddin*, Sixto Recavarren{dagger} and Jon S. Friedland2,*

* Department of Infectious Diseases and Wellcome Trust Centre for Clinical Tropical Medicine, Faculty of Medicine, Imperial College, Hammersmith Campus, London, United Kingdom; and {dagger} Universidad Peruana Cayetano Heredia and {ddagger} Asociacion Benefica Prisma, Lima, Peru


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tuberculosis is characterized by granuloma formation and caseous necrosis, but the factors causing tissue destruction are poorly understood. Matrix metalloproteinase (MMP)-9 (92-kDa gelatinase) secretion from monocytes is stimulated by Mycobacterium tuberculosis (M. tb) and associated with local tissue injury in tuberculosis patients. We demonstrate strong immunohistochemical MMP-9 staining in monocytic cells at the center of granuloma and adjacent to caseous necrosis in M. tb-infected patient lymph nodes. Minimal tissue inhibitor of MMPs-1 staining indicated that MMP-9 activity is unopposed. Because granulomas characteristically contain few mycobacteria, we investigated whether monocyte-monocyte cytokine networks amplify MMP-9 secretion. Conditioned medium from M. tb-infected primary human monocytes or THP-1 cells (CoMTB) stimulated MMP-9 gene expression and a >10-fold increase in MMP-9 secretion by monocytes at 3–4 days (p < 0.009, vs controls). Although CoMTB stimulated dose-dependent MMP-9 secretion, MMP-1 (52-kDa collagenase) was not induced. Anti-TNF-{alpha} Ab but not IL-1R antagonist pretreatment decreased CoMTB-induced MMP-9 secretion by 50% (p = 0.0001). Anti-TNF-{alpha} Ab also inhibited MMP-9 secretion from monocytic cells by 50%, 24 h after direct M. tb infection (p = 0.0002). Conversely, TNF-{alpha} directly stimulated dose-dependent MMP-9 secretion. Pertussis toxin inhibited CoMTB-induced MMP-9 secretion and enhanced the inhibitory effect of anti-TNF-{alpha} Ab (p = 0.05). Although chemokines bind to G protein-linked receptors, CXCL8, CXCL10, CCL2, and CCL5 did not stimulate monocyte MMP-9 secretion. However, the response to cholera toxin confirmed that G protein signaling pathways were intact. In summary, MMP-9 within tuberculous granuloma is associated with tissue destruction, and TNF-{alpha}, critical for antimycobacterial granuloma formation, is a key autocrine and paracrine regulator of MMP-9 secretion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tuberculosis (TB)3 is a global disease that kills 3,000,000 people each year, and resistance to standard antituberculous drugs is an escalating problem (1). In patients with TB, tissue destruction is a characteristic feature, and the histological hallmark of infection is caseous necrosis within antimycobacterial granuloma (2). Granulomas consist of densely compacted monocytic cells that contain the pathogen and are in turn surrounded by T lymphocytes. TNF-{alpha} is essential for granuloma development in mice infected with mycobacteria (3, 4, 5). Therapeutic TNF-{alpha} blockade for inflammatory pathologies is associated with reactivation of TB in patients (6). However, in addition to such protective immune responses, TNF-{alpha} may also be involved in the immunopathology of TB (4, 7). In particular, TNF-{alpha} is a major determinant of cerebral injury in a rabbit model of tuberculous meningitis (8), and TNF-{alpha}R p55-deficient mice, unable to neutralize TNF-{alpha}, developed fatal granulomatous necrosis following mycobacterial infection (9). Recently, secretion of TNF-{alpha} by human macrophages infected with Mycobacterium tuberculosis (M. tb) was shown to enhance intracellular multiplication of bacilli (10). Therefore, there appears to be a fine line between the protective and deleterious effects of TNF-{alpha}, which may determine the course of clinical disease. However, the precise factors responsible for tissue injury in TB remain poorly understood.

Matrix metalloproteinases (MMPs) form a family of zinc-containing proteases that degrade all extracellular matrix components and have a major role in physiological tissue remodeling and important immunomodulatory functions (11, 12). In particular, MMP-9 (92-kDa gelatinase) is the quantitatively predominant MMP secreted by monocytes, which are pivotal cells in the immune response to M. tb. MMP-9 facilitates leukocyte extravasation into infected sites by degrading type IV collagen in vascular basement membranes (13). In monocytic cells, MMP-9 secretion is tightly controlled at the level of gene transcription and may be up-regulated by cytokines, including TNF-{alpha} (14). Following secretion, MMPs are regulated by activation of latent proforms and by specific tissue inhibitors of MMPs (TIMPs), which form inhibitory complexes of 1:1 stoichiometry with MMPs. TIMP-1 is the major monocyte-derived TIMP, and the balance between the relative local concentrations of MMP and TIMPs determines net proteolytic activity (15). MMP-9 secretion by monocytic cells is potently stimulated by infection with M. tb. There is increasing evidence that, although physiological MMP-9 concentrations may be critical for leukocyte recruitment to control pathogen growth, unrestricted MMP-9 activity may contribute to host tissue injury in TB (16, 17, 18, 19, 20, 21). We have described a matrix-degrading phenotype in M. tb-exposed monocytes with increased secretion of MMP-9 but not TIMP-1. Furthermore, elevated MMP-9 activity (but not TIMP-1 levels) was found in cerebrospinal fluid from patients with tuberculous meningitis and was related to fatal outcome and cerebral injury (17). The fact that the signaling pathways regulating MMP-1/9 and TIMP-1 secretion are distinct in monocytic cells infected with M. tb, is a further indication of differential regulation of this enzyme and its principal inhibitor (19).

We now investigate the patterns of expression of MMP-9 and TIMP-1 within tuberculous granuloma and as a consequence explore the hypothesis that the cellular interactions between monocytes drive MMP-9 secretion in granuloma, where the numbers of mycobacteria tend to be low. Immunohistological analysis of M. tb-infected lymph nodes revealed intense, cytoplasmic MMP-9 staining of the majority of monocytic cells at the center of tuberculous granuloma and in areas adjacent to caseous necrosis. In contrast, there was sparse TIMP-1 staining, suggesting that MMP activity was unopposed. Investigating the mechanisms behind such extensive monocyte MMP-9 secretion, we show that, in M. tb-stimulated monocytes, TNF-{alpha} has a pivotal role in regulating MMP-9 gene expression, secretion, and gelatinolytic activity acting in both an autocrine and paracrine manner. Similar networking effects were not found to influence MMP-1 secretion. IL-1, a key cytokine in other pulmonary networks activated during host defense to M. tb (22), was not involved in driving MMP-9 secretion, whereas signaling via G protein-coupled receptors may be important.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Media, reagents, and Abs

For immunohistochemistry, mouse monoclonal anti-human MMP-9 Ab (clone VIIC2; Ab-8) and mouse monoclonal anti-human TIMP-1 Ab (clone 102D1; Ab-2) were obtained from Labvision (Fremont, CA). Bound primary Ab was detected using the Ultravision Detection System (catalog no. TM-015-HD), also purchased from Labvision. RPMI 1640 was obtained from Life Technologies (Paisley, U.K.), and Dubos enriched medium was obtained from Difco (Detroit, MI). rTNF-{alpha}, anti-TNF-{alpha} Ab, and IL-1R antagonist (IL-1Ra) were obtained from PeproTech (London, U.K.). Bordetella pertussis toxin and Vibrio cholera toxin (type Inaba 59B) were from Calbiochem (La Jolla, CA). For preparation of zymogram gels, AccuGel 29:1 (30% acrylamide, 29:1 acrylamide:bis-acrylamide), ProtoGel stacking and ProtoGel running buffers were purchased from National Diagnostics (Atlanta, GA). Triton X-100 was purchased from BDH (Poole, U.K.). Coomassie blue tablets were obtained from Pharmacia Biotech (Uppsala, Sweden). rMMP-9 (proform) was purchased from Calbiochem. Redi-Prime II random primer labeling system, [{alpha}-32P]dCTP, [{gamma}-32P]dCTP, nitrocellulose membranes (Hybond-N and Hybond-C), and Hyperfilm ECL were purchased from Amersham (Little Chalfont, U.K.). Kodak (Rochester, New York) Biomax MS-1 film was used for autoradiography. For Western blotting, sheep anti-human pro-MMP-9 Ab and peroxidase-conjugated donkey anti-sheep IgG were purchased from The Binding Site (Birmingham, U.K.). For the MMP-1 ELISA, HRP was obtained from DAKO (Glostrup, Denmark). All other reagents were purchased from Sigma-Aldrich (Poole, U.K.).

Immunohistological staining

Immunohistochemistry for MMP-9 and TIMP-1 was performed on paraffin-embedded lymph node sections from four patients with tuberculous lymphadenitis. Patients were diagnosed at Universidad Peruana Cayetano Heredia by positive culture of M. tb, and all were HIV-seronegative adults who had localized disease and responded well to conventional chemotherapy. All tissue was archived material obtained from biopsies performed for clinical indications. The ethical committees of Asociacion Benefica Prisma and Imperial College approved the use of these samples for this study. Sections were first dewaxed and then treated with 0.6% hydrogen peroxide to deplete endogenous peroxidase activity. Sections for TIMP-1, but not MMP-9, immunohistochemistry were boiled in 10 mM citrate buffer (pH 6.0) for 15 min followed by cooling at room temperature for 20 min. The sections were then blocked with 5% normal goat serum and, after washing in PBS, incubated at room temperature with either mouse monoclonal anti-human MMP-9 Ab (4–8 µg/ml) for 1 h or mouse monoclonal anti-human TIMP-1 Ab (1–2 µg/ml) for 30 min. After washing in buffer, the Ultravision Detection System was used to detect bound primary Ab. In brief, this involved sequential incubation with biotinylated goat anti-mouse IgG (1/500 dilution), enzyme-labeled streptavidin, and substrate chromagen. Human placenta and breast carcinoma (catalog no. MS-608-PCS; Labvision) were used as positive control tissue for MMP-9 (23) and TIMP-1 (24), respectively. In further negative control experiments, the biotinylated secondary Ab was omitted. All tissue sections were counterstained with H&E.

Culture of M. tb

Stocks of live, virulent M. tb (strain H37-Rv from Dr. J. Colston (National Institute of Medical Research, London, U.K.)) were maintained at 37°C in Dubos medium enriched with albumin Cohn fraction V plus dextrose and sodium chloride (endotoxin level, <3 pg/ml). To generate single-cell suspensions, aliquots were briefly sonicated and passed 10–12 times through a 22-gauge needle. This was confirmed by modified Kinyoun staining. The multiplicity of infection (MOI) used in experiments was quantitated by colony counting in triplicate on Middlebrook 7H10 plates (Difco).

Human monocytic cell culture

THP-1 cells (25) were obtained from the European Collection of Animal Cell Cultures (no. 88081201; Salisbury, U.K.) and maintained in RPMI 1640, supplemented with 10% FCS (endotoxin level, <20 pg/ml), L-glutamine (2 mM), and ampicillin (100 µg/ml) (ampicillin is not active against M. tb at this concentration). Human PBMCs were prepared from pooled buffy coats obtained from North Thames Blood Transfusion Service (Colindale, U.K.). PBMCs were isolated by density gradient centrifugation on Ficoll-Paque (Amersham Pharmacia, Little Chalfont, U.K.), and monocytes were purified by adhesion to tissue culture plastic for 2 h.

Experimental protocol

THP-1 cells or primary monocytes were suspended at a density of 2 x 106 cells/ml in standard 6-, 12-, or 24-well tissue-culture plastic plates. Cells were cultured in serum-free RPMI 1640, containing L-glutamine (2 mM) and ampicillin (100 µg/ml), and incubated in a humidified 5% CO2 atmosphere at 37°C. Human peripheral blood monocytes or THP-1 cells were infected with M. tb (MOI of 1), and the conditioned medium (CoMTB) was harvested at 24 h and stored at -20°C. Conditioned medium from unstimulated cells were negative controls (CoMControl). After preliminary dose response experiments, unstimulated monocytic cell cultures were exposed to CoMTB or CoMControl at a 1/5 dilution. To investigate cytokine-networking effects, CoMTB was preincubated with anti-TNF-{alpha} Ab (1–100 µg/ml) for 2 h at 37°C, and monocytic cells were preincubated with either IL-1Ra (1–100 ng/ml) or anti-TNF-{alpha} Ab (100 µg/ml) for 2 h. We previously confirmed in studies on chemokine biology that anti-TNF (50 µg/ml) neutralizes activity of 10 ng/ml TNF-{alpha} (22). In other experiments, THP-1 cells were directly stimulated with TNF-{alpha} (1 pg/ml to 10 ng/ml). To investigate the role of chemokine networks, THP-1 cells were preincubated with pertussis toxin (100 ng/ml) for 6 h before CoMTB stimulation or directly stimulated with cholera toxin (10 ng/ml), or with 100 ng/ml monocyte chemoattractant protein-1 (CCL2), RANTES (CCL5), IL-8 (CXCL8), and IFN-{gamma}-inducible protein-10 (CXCL10), either individually or in combination. Cellular RNA and/or culture supernatants from stimulated monocytic cells were harvested at 2–4 days and stored at -70 and -20°C, respectively, for further analysis. Cell viability was assessed by exclusion of trypan blue. All data are representative of experiments performed in triplicate on at least two separate occasions.

Gelatin zymography

MMP-9 was detected by zymography using standard methodology (26). Briefly, cell culture supernatants were mixed with 5x loading buffer (50 mM Tris-HCl (pH 7.6), 10% glycerol, 1% SDS, 0.01% bromophenol blue) and resolved on an 11% SDS gel impregnated with 0.12 mg/ml gelatin. Gels were run at 180 V for ~3 h, washed in 2.5% Triton X-100 for 1 h, and incubated in collagenase buffer (50 mM Tris-HCl (pH 7.6), 0.2 M NaCl, 5 mM CaCl2) at 37°C overnight. A working stain solution was prepared by diluting 20x 0.2% Coomassie blue stock solution with destain (1:3:6, glacial acetic acid:methanol:distilled water). After 2-h staining, proteolytic activity is revealed as white bands on a dark background. Gels were digitized using a transilluminator (UVP, Cambridge, U.K.), and densitometric analysis of proteolytic bands was performed using Image 1.61 analysis program (National Institutes of Health, Bethesda, MD). A linear range for quantitation of MMP-9 (6–170 ng/ml) was determined from standard curves obtained by running zymograms with known quantities of rMMP-9 (32–8000 pg). Samples containing MMP-9 activity above the upper limit of this range were diluted as necessary.

Western blot analysis

Western blot analysis was performed to demonstrate that the gelatinolytic bands on zymograms represented MMP-9 activity. Cell culture supernatants run on 10% SDS gels were transferred to Hybond-C and blocked (0.1% Tween 20 in PBS, 5% nonfat milk) for 1 h at room temperature. Blots were then incubated overnight with sheep anti-human pro-MMP-9 Ab (1:1000) at 4°C and, after washing, incubated with peroxidase-conjugated donkey anti-sheep IgG (1:1000) for 1 h at room temperature. Protein bands were visualized on Hyperfilm ECL by chemiluminescence.

Northern blotting and cDNA hybridization

RNA was extracted from 5 x 106 cells using a modified guanidium thiocyanate-phenol-chloroform method (27) with Tri-Reagent, according to the manufacturer’s instructions. Aliquots of RNA (12–15 µg) were run on denaturing 1% agarose-formaldehyde gels, transferred by capillary blotting to Hybond-N, and fixed by exposure to UV light (UV Stratalinker 1800; Stratagene, La Jolla, CA). The 0.5-kb cDNA MMP-9 probe (28) was a generous gift from Prof. H. Welgus (University of Washington, School of Medicine, St. Louis, MO) and was labeled with [{alpha}-32P]dCTP using a Redi-Prime II kit (random primer method). Following prehybridization, blots were hybridized overnight with radiolabeled probe, washed, and finally autoradiographed for 24–48 h at -70°C. Blots were stripped by heating for 1 h at 65°C in probe remover (0.005 M Tris-HCl (pH 8.0), 0.0002 M EDTA, 0.1x Denhardt’s solution) and then reprobed with {gamma}-32P-end-labeled, 42-mer {beta}-actin probe (29). Measurement of expression of this housekeeping gene plus assessment of 18/28S ribosomal RNA on agarose gels were used to confirm that loading of total RNA was uniform.

MMP-1 ELISA

MMP-1 was measured in cell culture supernatants using a previously described method (30), and ELISA reagents were a kind gift from Prof. T. Cawston (University of Newcastle, Newcastle-Upon-Tyne, U.K.). Briefly, standard 96-well plates were coated overnight at 4°C with RRU-CL1 mAb (1 µg/ml in PBS). After blocking with 10 mg/ml BSA in PBS for 1 h, MMP-1 standards and samples were loaded. After overnight incubation at 4°C, B-anti-CL1 biotinylated polyclonal Ab (2 µg/ml in PBS containing 0.5 mg/ml BSA and 0.1% Tween 20) was added for 2 h at room temperature. Binding was detected by addition of streptavidin HRP (1:5000) using o-phenylenediamine as a substrate. The reaction was stopped with 2 M H2SO4, and absorbency at 492 nm was measured. The lower limit of assay sensitivity is 5 ng/ml.

Statistics and data presentation

Results are expressed as mean ± SEM. Data are analyzed by unpaired t tests, and p < 0.05 was taken as significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
High-level MMP-9, but not TIMP-1, expression in monocytic cells within tuberculous granuloma

To determine whether MMP-9 activity could be detected in TB in vivo, sections from patients with tuberculous lymphadenitis were examined by immunohistochemistry. Fig. 1A shows that monocytes and macrophages, densely compacted at the center of the granuloma, stained strongly for MMP-9. It is striking that nearly every monocytic cell was positive for cytoplasmic MMP-9 staining. In comparison, there was weak staining in surrounding T lymphocytes and nongranulomatous lymphoid tissue. In marked contrast to MMP-9 staining in granuloma, only a few TIMP-1-positive stromal cells were identified throughout tissue sections (Fig. 1C), and TIMP-1 staining was absent in monocytic cells. Marked cellular staining for MMP-9, but not TIMP-1, was also found in the inflammatory infiltrate adjacent to areas of caseous necrosis (Fig. 1D) and within multinucleate giant cells (data not shown). No acid-fast mycobacteria were seen on Ziehl-Neelson staining, which suggests that the bacillary load was not very high, although this is not a sensitive technique. However, culture of biopsy specimens was not routine at diagnosis, and it was not possible to retrospectively culture formalin-fixed tissue.



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  		FIGURE 1. MMP-9 secreted by monocytic cells within tuberculous granuloma is unopposed by TIMP-1 and associated with tissue damage. A, Immunohistochemical analysis shows the majority of monocytes and macrophages, densely compacted at the center of the tuberculous granuloma, stain strongly for MMP-9. The surrounding layer of T lymphocytes is only weakly MMP-9 positive. B, Staining is absent in the negative, isotype-matched control. C, In contrast to MMP-9 staining, few TIMP-1-positive stromal cells are identified within granuloma. D, Strongly MMP-9-positive cells in inflammatory infiltrate surround an area of caseous necrosis. Counterstain is H&E. Magnification, x400 (A and B), x1000 (C), and x200 (D).

 
Monocyte-monocyte networks stimulate MMP-9, but not TIMP-1, secretion

We next investigated using zymography whether monocyte-monocyte networks might account for the high level of MMP-9 secretion within granuloma. At a 1/5 dilution, CoMTB, harvested from M. tb-infected primary monocytes at 24 h, strongly stimulated MMP-9 secretion (503 ± 92 ng/ml) from uninfected primary cells at 3 days, whereas CoMControl had a barely detectable effect (40 ± 29 ng/ml) (p < 0.009; Fig. 2A). CoMTB from M. tb-infected primary monocytes had an identical effect on THP-1 cells (278 ± 5 ng/ml) at 3 days, as did THP-1 cell-derived CoMTB on THP-1 cells (328 ± 9 ng/ml) at 4 days, compared with CoMControl (4 ± 0 ng/ml in both cases) (p < 0.0001; Fig. 2, B and C). Thus, CoMTB (derived from THP-1 or primary cells) stimulated a >10-fold increase in MMP-9 secretion by monocytic cells compared with controls. Furthermore, MMP-9 secretion was potently induced by CoMTB, because the MMP-9 concentration in each well at the zero time point (i.e., immediately after addition of CoMTB) was <30 ng/ml. MMP-9 secretion from monocytic cells exposed to CoMControl was not significantly different from basal release from unstimulated THP-1 cells. The findings were confirmed to be specific by Western blotting (Fig. 2D). These data further confirm previous studies that show that THP-1 cells are an appropriate monocyte model in which to study MMP-9 activity in models of TB (17, 18, 19, 20, 31). The addition of CoMTB to monocytic cells had a dose-dependent effect on MMP-9 secretion (Fig. 2E). MMP-1 is the second major macrophage-derived MMP whose secretion is stimulated by direct infection with M. tb (19). However, CoMTB (1/5 dilution) stimulation of THP-1 cells caused barely detectable secretion of MMP-1 (<10 ng/ml; ELISA data not shown).



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  		FIGURE 2. Monocyte-monocyte networks stimulate MMP-9 secretion. A, A representative zymogram with densitometric analysis show that CoMTB from M. tb-infected primary cells (1/5 dilution) stimulates MMP-9 secretion from uninfected primary cells at 3 days, whereas CoMControl had a barely detectable effect (p < 0.009). B and C, CoMTB from M. tb-infected primary cells (B) has an identical effect on THP-1 cells at 3 days as does CoMTB derived from M. tb-infected THP-1 cells (C) on THP-1 cells at 4 days (all p < 0.0001, compared with CoMControl). D, Western blot demonstrates MMP-9 secretion by M. tb-infected THP-1 cells but not unstimulated controls at 48 h, and that the 92-kDa gelatinolytic band visualized on zymograms represents MMP-9 activity. E, CoMTB stimulates dose-dependent secretion of MMP-9 by monocytic cells at 3 days. All data are representative of at least two independent experiments performed in triplicate. Bar charts show mean values ± SEM.

 
TNF-{alpha} has a pivotal role in monocyte-monocyte networks that stimulate MMP-9 secretion

We next investigated potential mediators in CoMTB, which might be responsible for inducing MMP-9 secretion from monocytic cells. Because TNF-{alpha} and IL-1 are major, monocyte-derived, proinflammatory mediators in TB (32, 33, 34, 35) that drive MMP-9 secretion by monocytes (18, 36, 37), we specifically investigated whether these cytokines were responsible for the activity of CoMTB. Pretreatment of CoMTB with anti-TNF-{alpha} Ab (100 µg/ml) reduced MMP-9 secretion by ~50% at 4 days compared with cells stimulated by CoMTB alone (p = 0.0001; Fig. 3A). Significant inhibition was not observed with anti-TNF-{alpha} Ab concentrations of <25 µg/ml. In addition, anti-TNF-{alpha} Ab (100 µg/ml) pretreatment markedly suppressed CoMTB-stimulated gene expression on Northern analysis (Fig. 3B). We next investigated whether there was a similar paracrine role for IL-1 by preincubating monocytic cells with human IL-1Ra (1–100 ng/ml) before stimulation with CoMTB. However, even 100 ng/ml IL-1Ra did not significantly alter CoMTB-induced MMP-9 secretion (p = 0.25), although there was a possible minor inhibitory trend (Fig. 3C). The range of biological activity of IL-1 Ra was 0.2–20 ng/ml, and the IL-1 concentration in undiluted CoMTB was <1 ng/ml. Importantly, neither TNF-{alpha} (100 ng/ml) nor IL-1Ra (100 ng/ml) significantly induced cell death or changed THP-1 cell proliferation in the time frame of these experiments.



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  		FIGURE 3. TNF-{alpha}, but not IL-1, is a critical stimulus of MMP-9 secretion involving monocyte-monocyte cytokine networks. A, Anti-TNF-{alpha} Ab (100 µg/ml) pretreatment inhibits CoMTB-induced MMP-9 secretion from monocytic cells by ~50% at 4 days (*, p = 0.0001). B, A representative zymogram and Northern blot shows that anti-TNF-{alpha} Ab (100 µg/ml) pretreatment reduces both the intensity of the proteolytic band produced by MMP-9 at 4 days and MMP-9 mRNA accumulation at 3 days. Expression of {beta}-actin mRNA, the housekeeping control gene, does not vary. C, CoMTB-induced MMP-9 secretion by THP-1 cells was not significantly reduced by human IL-1Ra preincubation (p = 0.25). All data are representative of at least three independent experiments performed in triplicate, and bar charts show mean values ± SEM.

 
Fig. 4A shows that TNF-{alpha} may directly stimulate MMP-9 secretion by monocytic cells in a dose-dependent manner. However, TNF-{alpha} alone stimulated less MMP-9 secretion than CoMTB (which contains <420 pg/ml TNF-{alpha}) and indicates that, although TNF-{alpha} is critical, other components of CoMTB may be required to enhance MMP-9 secretion. The zymogram in Fig. 4B confirms the specificity of the anti-TNF-{alpha} Ab used in these experiments. Anti-TNF-{alpha} Ab (100 µg/ml) completely blocked the direct effect of TNF-{alpha} (10 ng/ml) on MMP-9 secretion, which confirms that an excess of anti-TNF-{alpha} Ab was used to inhibit TNF-{alpha} activity in experiments using CoMTB.



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  		FIGURE 4. TNF-{alpha} directly stimulates MMP-9 secretion by monocytic cells. A, Densitometric analysis of an experiment performed in triplicate together with a representative zymogram demonstrate that TNF-{alpha} directly stimulates MMP-9 secretion by monocytic cells in a dose-dependent manner at 4 days. B, A zymogram demonstrates specificity of the anti-TNF-{alpha} Ab, because pretreatment with anti-TNF-{alpha} Ab (100 µg/ml) completely inhibits the direct effect of TNF-{alpha} (10 ng/ml) on MMP-9 secretion at 2 days. Data are all from at least two experiments performed in triplicate. Mean values ± SEM are shown.

 
Autocrine TNF-{alpha} induces MMP-9 gene expression and secretion from monocytic cells

We next investigated whether autologous TNF-{alpha} released from M. tb-infected cells might contribute to MMP-9 gene expression and secretion during direct infection. As reported previously (17), 2 days after infection with M. tb (MOI of 1), there is a 10-fold increase in MMP-9 secretion by THP-1 cells compared with controls (p < 0.0001). Preincubation with anti-TNF-{alpha} Ab (100 µg/ml) resulted in a 50% reduction in MMP-9 secretion, consistent with a significant autocrine TNF-{alpha} effect (p = 0.0002; Fig. 5A). In addition, mRNA accumulation in M. tb-infected monocytic cells was substantially reduced by anti-TNF-{alpha} Ab (Fig. 5B).



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  		FIGURE 5. Autocrine TNF-{alpha} release induces MMP-9 gene expression and secretion by monocytic cells. A, Densitometric analysis of an experiment performed in triplicate together with a representative zymogram demonstrating that 2 days after direct infection with M. tb (MOI of 1), there is a 10-fold increase in MMP-9 secretion by THP-1 cells (**, p < 0.0001, compared with controls), and anti-TNF-{alpha} Ab (100 µg/ml) pretreatment inhibits secretion by 50% (*, p = 0.0002). B, Northern analysis demonstrates that pretreatment with anti-TNF-{alpha} Ab (100 µg/ml) almost completely inhibits MMP-9 mRNA accumulation in M. tb-infected monocytic cells at 1 day compared with {beta}-actin. All data are representative from at least two experiments performed in triplicate. Mean values ± SEM are shown.

 
Pertussis toxin enhances the inhibitory effect of TNF-{alpha} blockade on CoMTB-induced MMP-9 secretion

Finally, because TNF-{alpha} seemed necessary but not sufficient to account for magnitude of networking effects, the potential role of chemokine networks in the context of infection by TB was investigated using pertussis toxin. This blocks intrinsic G protein function that couples transmembrane chemokine receptors with intracellular effectors and inhibits MMP-9 secretion (38). Pretreatment with pertussis toxin inhibited CoMTB-stimulated MMP-9 secretion by THP-1 cells in a dose-dependent manner, demonstrating a true biologic effect (Fig. 6A). In combination, anti-TNF-{alpha} Ab plus pertussis toxin treatment inhibited CoMTB-induced MMP-9 secretion more than TNF-{alpha} blockade alone, suggesting that chemokines may enhance the paracrine effects of TNF-{alpha} (Fig. 6B). However, although stimulation with cholera toxin confirmed that G protein-coupled signaling pathways were intact, direct exposure to CCL2, CCL5, CXCL8, and CXCL10 did not induce MMP-9 secretion in high concentration (100 ng/ml) at 4 days, either individually or in combination (data not shown).



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  		FIGURE 6. Pertussis toxin enhances the inhibitory effect of TNF-{alpha} blockade on CoMTB-induced MMP-9 secretion. A, Pertussis toxin pretreatment produces dose-dependent inhibition of CoMTB-induced MMP-9 secretion by THP-1 cells at 4 days. B, Pretreatment with either anti-TNF-{alpha} Ab (100 µg/ml) or pertussis toxin (10 ng/ml), significantly inhibits MMP-9 secretion by THP-1 cells in response to CoMTB at 4 days (p < 0.002 and p < 0.01, respectively, compared with cells exposed to CoMTB alone). However, combined treatment with anti-TNF-{alpha} Ab plus pertussis toxin inhibits CoMTB-induced MMP-9 secretion more than anti-TNF-{alpha} Ab alone (p = 0.05). Data are representative from at least two experiments performed in triplicate. Mean values ± SEM are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our data show for the first time that monocytic cells, closely packed together at the center of human tuberculous granuloma, exhibited strong cytoplasmic staining for MMP-9, but not TIMP-1. This raised the possibility that monocyte-monocyte cytokine networks might amplify MMP-9 secretion. In a cellular model, TNF-{alpha}, essential for granuloma formation and secreted by monocytic cells, was identified as a critical mediator of 92-kDa gelatinase secretion acting by autocrine and paracrine mechanisms. Thus, although MMP-9 and TNF-{alpha} have major and complementary roles in leukocyte recruitment and granuloma development necessary to restrict growth of M. tb, both may contribute to tissue injury during the immune response in TB.

This is the first study to investigate MMP and TIMP secretion in human tuberculous granuloma. Although previous studies had found that MMP-9 was up-regulated or associated with tissue damage in TB (16, 17, 18, 19, 20, 21), the extent to which MMP-9 expression was associated with nearly all monocytic cells in infected lymph nodes was unexpected. In contrast, the surrounding T cell population was much less densely stained, and this may reflect the fact that lymphocytes release less 92-kDa gelatinase than do monocytes. In addition, MMP-9 staining was prominent in the cellular infiltrate directly adjacent to areas of caseous necrosis consistent with the hypothesis that excessive MMP-9 secretion contributes to tissue destruction. MMPs have also been implicated in tissue injury in other human granulomatous disorders. For example, MMP-9 and TNF-{alpha} are coexpressed by macrophages in granuloma annulare (39), and MMP-mediated intestinal injury is inhibited by TNF-{alpha} blockade in Crohn’s disease (40). Because TIMP-1 cellular staining was sparse in tuberculous granuloma, the data suggest that monocyte-derived gelatinase activity is unopposed, producing a shift toward a matrix-degrading phenotype that we have described in TB patients (17). Although we previously found that TIMP-1 was constitutively secreted by monocytes and up-regulated following acute infection with M. tb in vitro (17), stromal cells, rather than monocytic cells within granuloma, appear to be the main source of this antiproteinase in M. tb-infected lymph nodes. The relative absence of TIMP-1 in vivo was therefore also unexpected, but it is possible that TIMP-1 secretion is down-regulated in more differentiated monocytic cells found in the established granuloma, which tend to be those that become necrotic. We are now investigating TIMP-1 secretion by fibroblasts, because these cells do appear to continue to secrete the inhibitor. Although it is possible that other TIMPs may regulate MMP-9 activity in granuloma, constitutive production of TIMP-2 by monocytic cells is decreased by cell stimulation (41), and TIMP-2 gene expression was reduced in the lungs of mice infected with M. tb (31).

The current histological data combined with our previous observation that M. tb at the very low MOI of 0.01 stimulates high-level MMP-9 secretion from monocytic cells (17), suggested that monocyte-monocyte cytokine networks amplify MMP-9 production within granuloma. This hypothesis is strongly supported by finding that CoMTB potently stimulated MMP-9 secretion from uninfected monocytes. Our findings that soluble TNF-{alpha} stimulated MMP-9 secretion and that specific TNF-{alpha} blockade decreased MMP-9 secretion by 50%, demonstrate that TNF-{alpha} is a major paracrine mediator. However, anti-TNF-{alpha} Ab activity appeared nonlinear, which may in part be due to concurrent nonspecific occupancy of FcRs. The fact that systemic TNF-{alpha} inhibition is associated with reactivation of human TB (6) reflects a central role for this cytokine in granuloma formation, whereas the intragranuloma TNF-{alpha} may be critical in influencing subsequent tissue destruction. In contrast to MMP-9, TNF-{alpha} alone is insufficient to stimulate MMP-1 secretion by monocytes (36). Interestingly, MMP-1 was not up-regulated by CoMTB, which suggests that the observed effect on MMP-9, the quantitatively most abundant monocyte-derived MMP, is relatively specific. In addition, our data suggest that an autocrine TNF-{alpha}-dependent loop mediates MMP-9 secretion by monocytic cells, and early TNF-{alpha} release by M. tb-infected monocytic cells may therefore induce MMP-9 gene transcription. Consistent with this is the fact that initiation of MMP-9 mRNA accumulation in monocytic cells following direct infection with M. tb is delayed (17). Despite the fact that some mice strains do not develop caseating granuloma in response to mycobacterial infection, data demonstrating that TNF-{alpha} blockade substantially reduced MMP-9 secretion from mouse peritoneal macrophages in response to live bacillus Calmette-Guérin infection, supports a similar central role for TNF-{alpha} (18).

It is likely that other mediators are also involved, because TNF-{alpha} blockade incompletely inhibited CoMTB-induced MMP-9 release, and concentrations of TNF-{alpha}, similar to those present in CoMTB, were less effective at stimulating MMP-9 secretion than CoMTB itself. Although IL-1 has a major role in human antimycobacterial immunity (42) and is known to selectively induce MMP-9 secretion by macrophages (36), our data show that IL-1 neutralization had no effect on CoMTB-stimulated MMP-9 release. Consistent with this, preincubation with an anti-IL-1{beta}R Ab did not reduce Mycobacterium bovis bacillus Calmette-Guérin-induced MMP-9 secretion by murine peritoneal macrophages (18). This contrasts with the importance of IL-1 in networks driving chemokine secretion in pulmonary TB (22). However, additional cytokines may be involved in the networking effect. For example, GM-CSF may enhance TNF-{alpha} or IL-1{beta}-stimulated MMP-9 secretion by THP-1 cells (37) and is expressed in human tuberculous granulomas (43). However, IFN-{gamma}, which synergizes with TNF-{alpha} to activate M. tb-infected macrophages and is essential for protective antimycobacterial immunity is not involved (44, 45). Our preliminary data show that M. tb-induced, monocyte-derived MMP-9 activity is down-regulated by IFN-{gamma}

Chemokines stimulate lymphocyte MMP-9 secretion (46), but little is known about the interactions between chemokines and MMPs in monocytic cells, and there are no data in the context of TB. However, G protein-linked pathways are involved in MMP-9 secretion by monocytic cells, and chemokine receptors are coupled to G proteins (38). Our primary aim was to examine whether G protein-linked pathways were involved in monocyte-monocyte networks in TB. The dose-response effect of pertussis toxin demonstrates a true biologic role for G proteins. We next investigated the effect of simultaneous TNF-{alpha} blockade and G protein inhibition, because recent work showed autologous TNF-{alpha} was required for chemokine-induced MMP-9 release by monocytic cells (47). Our data demonstrate that there is significant further down-regulation of MMP-9 secretion in the presence of both anti-TNF-{alpha} Ab and pertussis toxin, compared with pretreatment with anti-TNF-{alpha} alone. However, MMP-9 was not secreted in direct response to several chemokines, CCL2, CCL5, CXCL8, and CXCL10, which are involved in the immune response in human TB (48, 49). Other chemokines may be involved, or alternatively, chemokine-independent G protein-mediated pathways may regulate MMP-9 signal transduction (36).

In summary, these data demonstrate that MMP-9 is strongly expressed in monocytes within tuberculous granuloma and is associated with tissue necrosis in vivo. TIMP-1 production is minimal. TNF-{alpha} is a pivotal mediator of monocyte-derived MMP-9 secretion acting by autocrine and paracrine mechanisms. G protein-linked pathways are involved in monocyte networks, but IL-1{beta} is not. Similar to TNF-{alpha}, the extent of MMP-9 activity within granuloma may be a critical factor determining the balance between control of infection and tissue destruction. Because excessive MMP-9 activity may result in tissue injury, synthetic MMP inhibitors may have a future role in the management of TB. Such drugs have been safely evaluated in clinical studies (50) and also decrease the concentration of soluble TNF-{alpha} by inhibiting TNF-{alpha}-converting enzyme, a closely related metalloenzyme that cleaves active TNF-{alpha} from its membrane-bound precursor (51). Recently, combined metalloproteinase/TNF-{alpha}-converting enzyme inhibition reduced levels of MMP-9, TNF-{alpha}, and cerebral injury in a rodent model of bacterial meningitis (52), and such novel therapeutic strategies now need to be investigated in animal models of TB.


   Acknowledgments
 
Dr. Dan Remick kindly performed TNF-{alpha} and IL-1 ELISA measurements. Prof. Gordon Stamp, Dr. Rob Hasserjian, Donna Horncastle, and Susan Van Noorden provided invaluable assistance with immunohistochemistry. Dr. Robert Wilkinson kindly read and commented on the manuscript.


   Footnotes
 
1 This work was funded by the Medical Research Council (United Kingdom). N.M.P. was supported by a Medical Research Council Clinical Training Fellowship. Back

2 Address correspondence and reprint requests to Dr. Jon S. Friedland, Department of Infectious Diseases, Faculty of Medicine, Imperial College, Hammersmith Campus, Du Cane Road, London, W12 0NN, U.K. E-mail address: j.friedland{at}imperial.ac.uk Back

3 Abbreviations used in this paper: TB, tuberculosis; M. tb, Mycobacterium tuberculosis; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of MMPs; IL-1Ra, IL-1R antagonist; MOI, multiplicity of infection; CoMTB, conditioned medium from M. tb-infected monocytes; CoMControl, conditioned medium from unstimulated control monocytes. Back

Received for publication March 31, 2003. Accepted for publication September 12, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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