Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0806512 on November 1, 2006

Published online before print November 1, 2006
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(Journal of Leukocyte Biology. 2007;81:548-556.)
© 2007 by Society for Leukocyte Biology

Monocytes infected with Mycobacterium tuberculosis regulate MAP kinase-dependent astrocyte MMP-9 secretion

James E. Harris, Justin A. Green, Paul T. Elkington and Jon S. Friedland1

Department of Infectious Diseases and Immunity, Imperial College, Hammersmith Campus, London, UK

1 Correspondence: Department of Infectious Diseases and Immunity, Imperial College, Hammersmith Campus, Du Cane Road, London, W12 0NN, UK. E-mail: j.friedland{at}imperial.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tuberculosis (TB) of the CNS (CNS-TB) carries a high mortality. Disease pathology is characterized by widespread destruction of CNS tissues. Matrix metalloproteinase-9 (MMP-9) is able to catabolyze specific components of the CNS tissue matrix and blood-brain barrier. Increased cerebrospinal fluid MMP-9 concentrations are associated with tissue damage, leukocyte infiltration, and death in CNS-TB. Using zymography, Western analysis, and transcription factor assays, we investigated mechanisms regulating MMP-9 activity in CNS-TB. We demonstrate that conditioned media from monocytes infected with Mycobacterium tuberculosis (CoMTB) induce MMP-9 secretion from astrocytes (U373-MG). IL-1ß and TNF-{alpha} are necessary but not sufficient for such induction of astrocyte MMP-9 secretion. CoMTB up-regulates AP-1 DNA-binding activity, and the c-Jun, FosB, and JunB subunits are particularly increased. MMP-9 secretion from CoMTB-stimulated astrocytes is dependent on the activity of p38, Erk, and Jnk MAPKs. Phosphorylation of p38, Erk, and Jnk is activated rapidly, peaking 30 min poststimulation with CoMTB. Inhibition of IL-1ß but not TNF-{alpha} in CoMTB decreases p38, Erk, and Jnk activity in astrocytes. Consistently, IL-1ß signals through the MAPK cascade at physiological levels, whereas TNF-{alpha}, IL-6, IL-10, CCL-2, CCL-5, and CXCL-8 (all present in CoMTB) do not. In summary, the data suggest that monocyte-dependent cytokine networks may play a key role in the development of a matrix-degrading environment during CNS-TB.

Key Words: gelatinase B • IL-1 • TNF-{alpha}


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mycobacterium tuberculosis is a highly successful pulmonary pathogen, able to manipulate host immune responses and persist in the lungs for whole lifetimes without causing disease [1 2 3 4 ]. In contrast, in the CNS, where the function of neurones is protected by the maintenance of an anti-inflammatory environment [5 ], infection with M. tuberculosis leads to catastrophic, inflammatory tissue destruction [6 ]. This is more severe than in other bacterial or viral meningitides [7 ]. For M. tuberculosis, invasion of the CNS represents an evolutionary dead-end, which without therapy, will almost certainly result in the death of the host [8 ]. The mechanisms behind this phenomenon are currently unclear, but better understanding of the pathogenesis of CNS-tuberculosis (TB) is required if improvements are to be made on therapies that still leave over half of those affected dead or paralyzed [9 ].

Matrix metalloproteinases (MMPs) have been implicated in inflammatory tissue destruction in a range of pathological situations in the CNS, including experimental autoimmune encephalomyelitis, multiple sclerosis, and CNS-TB [6 , 7 , 10 , 11 ]. Together, MMPs are able to catabolyze all components of the extracellular matrix, thus possessing unlimited potential to damage CNS tissues [12 , 13 ]. Excessive MMP-9 activity is associated with damage to neurones and CNS tissues [14 ]. MMP-9 may disrupt the blood-brain barrier (BBB) as a result of degradation of collagen type IV in the basal-lamina and may also break down many CNS matrix components [15 ]. MMP-9 knockout mice are protected against ischemic and post-traumatic damage, which follow BBB disruption [16 ]. In CNS-TB, we found that increased MMP-9 secretion is relatively unopposed by a corresponding rise in tissue inhibitor of MMP-1 concentrations, resulting in a matrix-degrading phenotype [6 ]. Elevated MMP-9 activity in cerebrospinal fluid (CSF) from patients with CNS-TB was associated with signs of local tissue destruction and death.

During the development of CNS-TB, peripheral mononuclear phagocytes infiltrate the CNS in large numbers [17 ]. Monocytes stimulated with M. tuberculosis secrete a broad range of cytokines including CCL-2, CXCL-8, IL-1ß, IL-6, and TNF-{alpha} [18 , 19 ]. IL-1ß and TNF-{alpha} have been found at increased concentrations in the CSF of patients with CNS-TB [20 21 22 ]. In pulmonary TB, monocyte-derived TNF-{alpha} increases MMP-1 and -9 secretion from pulmonary epithelial cells [18 , 23 ]. However, the effect of infiltrating monocytes on MMP secretion by CNS-resident cells during CNS-TB is unknown.

Astrocytes are the most populous cell type in the CNS, outnumbering neurones by a factor of 10 [24 ]. Their role in the maintenance of homeostasis and a noninflammatory environment in the normal CNS is well documented [25 ]. However, the role that astrocytes play in control of MMP-9 secretion in CNS-TB is unknown. Monocyte activation and interaction with astrocytes have a central role in the development of pathology in inflammatory and infectious diseases of the CNS including HIV-associated dementia and neurocysticercosis [26 , 27 ], and astrocyte MMP secretion is induced by a number of cytokines secreted by monocytes. For example, MMP-9 secretion from rat astrocytes is up-regulated in response to IL-1ß and TNF-{alpha} [28 , 29 ]. Interactions between monocyte-derived cytokines and astrocytes and their role in the control of MMP secretion during CNS-TB have not been investigated.

In normal physiology, expression of MMPs is controlled tightly by transcriptional and translational mechanisms [30 ]. There are multiple transcription factor-binding sites for AP-1 in the MMP-9 promoter [31 ]. Although AP-1 and NF-{kappa}B activation may be involved in maximal up-regulation of MMP-9 [32 ], AP-1 is a critical regulator in the control of MMP-9 secretion in response to TNF-{alpha} and IL-1ß [33 ]. AP-1 consists of homodimers of the Jun family (c-Jun, JunB, and JunD) or heterodimers of Jun and Fos family proteins (c-Fos, FosB, Fra-1, and Fra-2). Different AP-1 dimers have subtly differing activities, which vary, depending on cell type and stimuli. Generally, c-Jun, c-Fos, and FosB are strong transactivators, whereas JunB, JunD, Fra-1, and Fra-2 are weaker transactivators, which may inhibit AP-1 by forming heterodimers of reduced activity with c-Jun, c-Fos, and FosB [34 ]. However, in fibroblasts, complexes of c-Jun with Fra-1 and -2 have similar activity to dimers of c-Jun and c-Fos [35 ]. The role of AP-1 in regulation of astrocyte MMP-9 secretion in CNS-TB is unknown.

The DNA-binding activity of AP-1 dimers is controlled in part via the MAPK. For example, c-Jun and c-Fos contain binding sites for JNK and ERK, respectively, in close proximity to their transactivation domains [36 , 37 ]. TNF-{alpha} and IL-1ß may signal through the MAPK and AP-1 pathways [28 , 38 ]. p38 MAPK mediates an increase of MMP-1 in a cellular model TB [18 ]. Involvement of MAPK cascades in regulation of astrocyte MMP responses to M. tuberculosis has not been investigated. In this study, we investigated the hypothesis that in CNS-TB, monocyte-dependent cytokine networks regulate MMP-9 secretion from astrocytes and examined the role of MAPK and AP-1 signaling pathways.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
All reagents were purchased from Sigma-Aldrich (Poole, UK), except for those listed below. For preparation of zymogram gels, AccuGel 29:1 (40% 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 VWR (Poole, UK). Coomassie blue tablets were obtained from Pharmacia Biotech (Uppsala, Sweden). For Western blotting, sheep antihuman pro-MMP-9 antibody and peroxidase-conjugated donkey antisheep IgG were purchased from The Binding Site (Birmingham, UK). For blocking experiments, goat antihuman TNF-{alpha} antibodies, rabbit antihuman IL-6 antibodies, and human IL-1 receptor antagonist (IL-1Ra) were purchased from Peprotech (London, UK). Pertussis toxin (PTX) and cholera toxin and the JNK, ERK, and p38 antagonists SP600125, PD98059, and SB203580, respectively, were purchased from Merck Biosciences (Nottingham, UK). Rabbit antibodies against phosphorylated and nonphosphorylated forms of human JNK, ERK, and p38, as well as peroxidase-conjugated antirabbit IgG were purchased from Cell Signaling Technology (Beverly, MA).

M. tuberculosis culture
M. tuberculosis H37-Rv was maintained in Middlebrook 7H9 medium supplemented with 10% albumin-dextrose-catalase enrichment medium, 0.2% glycerol, 0.02% Tween-80, and 2.5 µg/ml amphotericin with agitation (BD Biosciences, San Jose, CA). M. tuberculosis was used at mid-log growth phase at OD ~0.60 (Biowave Cell Density Meter, WPA, Cambridge, UK) in all experiments. Clump-free mycobacterial suspensions were produced by sonicating stock cultures of M. tuberculosis for 30 s immediately prior to use. M. tuberculosis endotoxin level was measured by the amoebocyte lysate assay (Associates of Cape Cod, East Falmouth, MA) and was less than 0.3 ng/ml LPS.

Cell culture and stimulation
Human astrocyte cell lines U373-MG and U87-MG (ECACC Nos. 89081403 and 89081402, respectively) were maintained in minimum essential medium Eagle (MEME), supplemented with 10% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, 1% nonessential amino acids, and 100 µg/ml ampicillin, according to supplier’s instructions. All experiments were performed in serum-free medium within 20 passages of revival.

Primary human blood monocytes were prepared from single-donor buffy coat residues obtained from healthy donors (National Blood Transfusion Service, UK). After density gradient centrifugation (Ficoll Paque, Amersham Biosciences, UK) followed by adhesion purification, monocyte purity was over 95% by FACS analysis. Monocytes were plated out at 250,000 cells/cm2 in RPMI with 2 mM glutamine and 10 µg/ml ampicillin and infected with M. tuberculosis at a multiplicity of infection (MOI) of 1. After incubation at 37°C for 24 h, conditioned medium was harvested, followed by filtration through a 0.2-µm Anopore membrane to remove M. tuberculosis. Conditioned media from monocytes infected with M. tuberculosis were termed CoMTB, and media from uninfected monocytes were CoMCon. CoTB was produced by incubating M. tuberculosis in RPMI (with 2 mM glutamine and 10 mg/ml ampicillin) for 24 h in the absence of monocytes and subsequently, processed identically to CoMTB and CoMCon.

Once confluent, U373-MG or U87-MG cells were stimulated with a 1:5 dilution of CoMTB, CoMCon, or CoTB:MEME, unless otherwise stated. CoMCon was run in parallel with CoMTB in all experiments. Supernatant was harvested from astrocytes at 72 h and centrifuged at 12,000 relative centrifugal force (RCF) for 5 min to remove cellular debris and was frozen immediately.

Cytokine bead array (CBA)
The BD PharMingen (San Diego, CA) human inflammation kit was used to analyze TNF-{alpha}, IL-1ß, IL-6, IL-10, IL-12 p70, and CXCL-8. The BD PharMingen human chemokine kit was used to measure CCL-2, CCL-5, CXCL-8, CXCL-9, and CXCL-10. A series of spectrally discrete particles captures the analytes, and the level is calculated by cytometric analysis on a FACSCaliber (Becton Dickinson, Franklin Lakes, NJ). This methodology is able to analyze multiple variables within a single sample simultaneously and generates data that is equivalent to an ELISA-based assay.

Zymography
Standard methodology for gelatin zymography was used to detect MMP-9 activity in samples [39 ]. In brief, standards and prepared cell supernatants were loaded with 5x loading buffer (0.25 M Tris, pH 6.8, 50% glycerol, 5% SDS, bromophenol blue) and run on 11% acrylamide gels impregnated with 0.1% gelatin as substrate. After ~3.5 h at 180 V (buffer 25 mM Tris, 190 mM glycine, 0.1% SDS), the gel was renatured in 2.5% Triton X for 1 h with gentle agitation at room temperature. After being washed twice in collagenase buffer (55 mM Tris base, 200 mM sodium chloride, 5 mM calcium chloride, 0.02% Brij, pH 7.6), gels were incubated for 16 h in fresh collagenase buffer at 37°C. Gelatinolytic activity was detected using 0.02% Coomasie blue in 1:3:6, acetic acid:methanol:water. All experimental samples were run in parallel with 20 pg per well recombinant MMP-9 (Oncogene, Cambridge, MA) to standardize between gels and to assist with identification of MMP-9. Gel images were photographed with a Trans-illuminator (UVP, Upland, CA) followed by proteolytic band quantification using LabWorks (Version 4.5). The digitized results of each sample were normalized to the standards. For EDTA inhibition of MMP activity, 10 mM EDTA was added to the collagenase buffer before 16 h inhibition.

Western blot analysis for detection of MMP-9 and MAPK phosphorylation
Western blotting was used to confirm MMP-9 secretion. After mixing 40 µl prepared cell supernatants with 2x loading buffer (10% glycerol, 5% 2-ME, 2% SDS, 0.06 M Tris, pH 6.8, bromophenol blue), each sample was heat-denatured and run on a 10% acrylamide gel at 200 V (running buffer 25 mM Tris base, 192 mM glycine, 0.1% SDS) for 3 h. After separation, proteins were transferred onto a nitrocellulose membrane (GE Healthcare, Little Chalfont, UK) and blocked for 1 h with 5% BSA/0.1% Tween-20. Membranes were then incubated with primary anti-MMP-9 antibody (one in 1000 dilution) at 4°C for 16 h. After washing, the membrane was incubated with peroxidase-conjugated secondary antibody (1:1000 dilution) for 1 h. Protein bands were visualized on Hyperfilm ECL using chemiluminescence.

To investigate phosphorylation of the MAPKs JNK, p38, and ERK, confluent cells were stimulated with conditioned media and incubated until a specific time-point. Cells were then washed with ice-cold PBS and scraped into ice-cold SDS sample buffer (62.5 mM Tris/2% SDS/10% glycerol/50 mM DL-dithiothreitol/0.01% bromophenol blue). Samples were frozen at –70°C until required. Each sample (40 µl) was processed as described above, except that MAPK-specific phospho- and total rabbit antihuman antibodies were used for probing Western blots.

Preparation of nuclear extracts
The NE-PER extraction kit (Pierce Biotechnology, UK) was used to produce nuclear and cytoplasmic extracts according to the manufacturer’s instructions. In brief, confluent cells were stimulated with conditioned media and incubated until the specified time-point. Cells were scraped into ice-cold 1x PBS and spun at 100 RCF to produce a cell pellet, which was resuspended in cold cytoplasmic extraction reagent (CER)1 containing Halt protease inhibitors (Pierce Biotechnology). Following 10 min incubation on ice, CER2 was added to break down the cytoplasmic membrane. After centrifugation (16,000 RCF), cytoplasmic extract was harvested and frozen immediately. Nuclear extraction reagent (NER) was added to the remaining nuclear pellet; after a 40-min incubation and centrifugation (16,000 RCF), the nuclear extract was harvested and frozen.

AP-1 nuclear-binding assay
Activation of the complex binding activity of the multiple subunits of AP-1 was analyzed using an ELISA-based assay (TransAMTM, Active Motif North America, Carlsbad, CA), which is five times more sensitive than EMSA. Nuclear extracts were added to a 96-well plate containing immobilized oligonucleotides encoding an AP-1 consensus site [5'-TGA(C/G)TCA-3']. Active AP-1-binding subunits contained in the nuclear extract bind specifically to this oligonucleotide. The primary antibodies used to detect c-Fos, c-Jun, FosB, Fra-1, Fra-2, JunB, or JunD recognize an epitope accessible only when the active form of these factors is bound to its target DNA. A HRP-linked secondary antibody was added, and the color change was determined by spectrophotometry at 450 nm. Competition experiments demonstrated specificity of binding by adding 20 pmol/well wild-type or mutated NF-{kappa}B oligonucleotide before assaying with the p65 antibody

Data presentation and statistical analysis
Data are presented as means ± SD of three samples and represent experiments performed in triplicate on at least two occasions, unless otherwise stated. Statistical analysis was performed using SPSS, Version 13.0 (SPSS Inc., Chicago, IL). Paired groups were compared with the Student’s t-test. Multiple intervention experiments were compared with one-way ANOVA followed by Tukey’s multiple comparison. A P value of <0.05 was taken as statistically significant. For all graphs, * represents a P value <0.05; ** represents a P value <0.01.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CoMTB causes a dose-dependent increase in astrocyte MMP-9 secretion
First, the effect of CoMTB on MMP-9 secretion from astrocytes was investigated. Stimulation of U373-MG and U87-MG cells with a 1:3 dilution of CoMTB induced MMP-9 secretion at 72 h (P<0.01; Fig. 1A and 1B , and data not shown). Even at a 1:20 dilution of CoMTB:MEME, significant up-regulation of MMP-9 secretion occurred (P<0.05). There was a nonsignificant increase in MMP-9 secretion at a 1:50 dilution of CoMTB, which does not contain detectable levels of MMP-9. Incubation of the zymogram in 10 mM EDTA abolished MMP-9 bands (Fig. 1A) , which together with Western analysis (data not shown), confirmed that enzymatic activity was a result of MMP-9. We also found that direct infection by M. tuberculosis at a MOI of 10 does not up-regulate MMP-9 secretion from astrocytes (Fig. 1C) . In addition, condition media from astrocytes stimulated with M. tuberculosis did not drive monocyte MMP-9 secretion (data not shown).


Figure 1
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  		Figure 1. Monocyte-derived stimuli up-regulate astrocyte MMP-9 in response to M. tuberculosis. (A) Representative zymograms showing dose-dependent secretion of MMP-9 in response to stimulation with CoMTB. (B) Densitometric analysis of zymography. U373-MG cells were stimulated with decreasing dilutions of CoMTB in MEME for 72 h. Control (Con) represents MMP-9 secretion from CoMCon-stimulated astrocytes at 72 h. (C) Densitometric analysis of representative zymography showing astrocyte MMP-9 secretion in response to CoMTB and M. tuberculosis (M.tb; MOI 10); control represents no stimulation. Bars on charts represent mean ± SD of two independent experiments performed in triplicate.

 
IL-1ß and TNF-{alpha} are necessary but not sufficient for CoMTB-induced MMP-9 secretion
To investigate the factors that may be active in CoMTB-mediated up-regulation of astrocyte MMP-9 secretion, blocking experiments were performed (Fig. 2A ). Preincubation astrocytes with 100 ng/ml IL-1Ra for 2 h or preincubation of CoMTB with 5 µg/ml anti-TNF-{alpha} for 2 h inhibited MMP-9 secretion in response to CoMTB at 72 h by 52 ± 17% (P<0.05) and 65 ± 10% (P<0.01), respectively. When these inhibitors were used in combination, there appeared to be an additive, inhibitory effect on MMP-9 secretion. However, this cumulative effect did not achieve statistical significance. To determine whether chemokines might also be driving astrocyte MMP-9 secretion, as reported for T lymphocytes [40 ], the effect of PTX, which inhibits signaling through G-protein-linked processes, was investigated. Pertussis (10 ng/ml) had no effect on the secretion of MMP-9 in response to CoMTB alone or in combination with 5 µg/ml anti-TNF-{alpha} and/or 100 ng/ml IL-1Ra. Anti-IL-6 was also shown to have no effect on CoMTB-induced MMP-9 secretion from astrocytes (data not shown).


Figure 2
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  		Figure 2. IL-1 and TNF-{alpha} regulate MMP-9 secretion from astrocytes. (A) Changes in secretion of MMP-9 from CoMTB-stimulated astrocytes following incubation with anti-TNF-{alpha} (5 µg/ml), IL-1Ra (100 ng/ml), or PTX (10 ng/ml) alone and in combination. (B) Astrocyte MMP-9 secretion induced by supraphysiological TNF-{alpha} and IL-1ß concentrations. (C) Comparison of MMP-9 secretion from CoMTB-stimulated astrocytes and astrocytes stimulated with TNF-{alpha} or IL-1ß (at concentrations found in CoMTB) or cholera toxin, alone and in combination. (D) Comparison of MMP-9 secretion from control, unstimulated astrocytes and astrocytes stimulated with CoMTB or CoTB. (E) MMP-9 secretion is not induced in astrocytes stimulated with M. tuberculosis, TNF-{alpha}, or IL-1ß alone or in combination. Data presented are densitometric analyses of gelatin zymography from triplicate experiments performed on two occasions.

 
To further examine the role of IL-1ß and TNF-{alpha} in CoMTB-dependent MMP-9 secretion, astrocytes were stimulated with these cytokines. MMP-9 secretion from astrocytes was inducible using supraphysiological levels of IL-1ß (50 ng/ml) and TNF-{alpha} (100 ng/ml; Fig. 2B ). However, CBA array analysis showed that TNF-{alpha} and IL-1ß are present in CoMTB at 0.78 ng/ml and 4.57 ng/ml, respectively. At the concentrations found in CoMTB, these cytokines did not generate MMP-9 secretion from astrocytes (Fig. 2C) . Cholera toxin, which stimulates G-protein-linked signaling pathways (including those activated by chemokine receptor binding), did not induce astrocyte MMP-9 secretion alone or in combination with IL-1ß and/or TNF-{alpha} (Fig. 2C) . We also investigated the potential role of antigens from M. tuberculosis in MMP-9 secretion from astrocytes (Fig. 2D) . Stimulation of astrocytes with cell-free conditioned media from M. tuberculosis (CoTB) induced no MMP-9 secretion from astrocytes. We next examined whether M. tuberculosis-derived stimuli might interact with IL-1ß and/or TNF-{alpha} to induce MMP-9 secretion from astrocytes (Fig. 2E) . IL-1ß, TNF-{alpha}, and/or M. tuberculosis did not induce MMP-9 secretion alone or in combination. CoMTB contains Oncostatin M (OSM), and previous studies have demonstrated MMP-inducing synergy between OSM and IL-1 [41 ]. However, we found that OSM had no effect on MMP-9 secretion from IL-1-stimulated astrocytes (data not shown).

CoMTB activates specific AP-1 subunits
Next, activation of AP-1 subunit DNA-binding activity in response to CoMTB was assayed (Fig. 3 ). CoMTB significantly up-regulates the DNA-binding activity of c-Jun, FosB, Fra-1, Fra-2, and JunB. JunB and FosB activity was increased by 81 ± 16% and 68 ± 12%, respectively (P<0.05), whereas Fra-1 and Fra-2 activity was increased by approximately 50% (P<0.05). The binding activity of c-Jun increased by 30 ± 5% in response to CoMTB stimulation (P<0.05). c-Fos activity was increased by 150 ± 108%, but this failed to achieve statistical significance, owing to the variable nature of this response. JunD activity was not increased by CoMTB stimulation. Specificity of the assay was confirmed by inhibition of binding using 20 pmol/well wild-type consensus oligonucleotide. A mutated consensus had no effect on AP-1 binding. The WI-38 (TPA+CI) nuclear extract was used as a positive control.


Figure 3
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  		Figure 3. AP-1 DNA-binding activity in response to CoMTB. Nuclear extracts from astrocytes (stimulated with CoMCon or CoMTB) are assayed for c-Fos, FosB, Fra-1, Fra-2, c-Jun, JunB, and JunD activity using the TransAM AP-1 family kit. The y-axis represents percentage increase in DNA-binding activity. Each bar is mean ± SD of data from three independent experiments.

 
MAPK signaling and CoMTB induced MMP-9 secretion from astrocytes
As MAPKs are important in the activation of the AP-1 subunit DNA-binding activity [33 ], we investigated whether MAPKs regulate CoMTB-stimulated pathways, resulting in astrocyte MMP-9 secretion. Inhibition of the p38 and ERK MAPK signaling pathways with the specific inhibitors SB203580 and PD98059, respectively [42 43 44 ], resulted in the near-complete inhibition of CoMTB-mediated MMP-9 secretion from astrocytes in a dose-dependent manner (Fig. 4 ). Inhibition of the JNK MAPK signaling pathway with the inhibitor SP600125, which exhibits over 300-fold greater selectivity for JNK as compared with ERK-1 and p38 MAPKs [45 ], also resulted in the near-complete inhibition of CoMTB-mediated MMP-9 secretion from astrocytes. Trypan blue exclusion showed no effect on cell viability in cultures treated with the inhibitors at the concentrations used.


Figure 4
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  		Figure 4. The effect of inhibiting MAPK activity on CoMTB-mediated secretion of MMP-9 by astrocytes. (A) Inhibition of the p38 pathway with SB203580 at 1, 10, and 30 µM. (B) Inhibition of ERK phosphorylation with PD98059 at 1, 10, and 30 µM. (C) Inhibition of JNK phosphorylation with SP600125 at 1, 5, and 10 µM. Data presented are a densitometric analysis of gelatin zymography of experiments repeated in triplicate on three occasions.

 
Maximal phosphorylation of all three MAPKs occurred at 30 min poststimulation with CoMTB and remained above baseline 4 h poststimulation (Fig. 5 ). p38 and JNK are not constitutively phosphorylated in U373-MG cells, and their phosphorylation was strongly induced by CoMTB but not by CoMCon (Fig. 5A and 5C) . ERK is constitutively phosphorylated in this astrocyte line. However, increased phosphorylation is detectable at 30 min in CoMTB- but not CoMCon-stimulated cells (Fig. 5B) . Levels of total p38, ERK, and JNK did not change during experiments providing supporting data that the inhibitors do not compromise cell viability at the concentrations used.


Figure 5
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  		Figure 5. MAPK phosphorylation kinetics in response to CoMTB and CoMCon. Representative Western blots from three separate experiments show kinetics of astrocyte MAPK phosphorylation following stimulation with CoMTB or CoMCon. (A) Total and phosphorylated p38; (B) total and phosphorylated ERK; (C) total and phosphorylated JNK.

 
IL-1Ra but not anti-TNF-{alpha} inhibits CoMTB-induced MAPK activation
To determine whether IL-1ß and TNF-{alpha} in CoMTB might up-regulate MMP-9 secretion via the MAPK pathway, we investigated the effect of inhibiting these cytokines on CoMTB-stimulated astrocyte MAPK phosphorylation (Fig. 6A 6B 6C ). Inhibition of IL-1 signaling with 100 ng/ml IL-1Ra reduced the phosphorylation of all three MAPKs, although their phosphorylation was greater than in control cells. In contrast, 5 µg/ml anti-TNF-{alpha} did not affect phosphorylation of any MAPK, despite decreasing MMP-9 secretion. Combined inhibition with IL-1Ra and anti-TNF-{alpha} had no additional or synergistic effect.


Figure 6
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  		Figure 6. CoMTB-mediated MAPK phosphorylation is dependent on IL-1. The effect of anti-TNF-{alpha} (5 µg/ml) and/or IL-1Ra (100 ng/ml) blockade of CoMTB on the phosphorylation of (A) p38, (B) ERK, or (C) JNK. Western blots are representative of three independent experiments showing the effects of stimulation with CoMTB or CoMTB-equivalent levels of CCL-2 (1 ng/ml), CCL-5 (355 pg/ml), CXCL-8 (11 ng/ml), IL-1ß (4.6 ng/ml), IL-6 (5 ng/ml), IL-10 (98 pg/ml), and TNF-{alpha} (380 pg/ml), alone or in combination, on the phosphorylation of (D) p38, (E) ERK, and (F) JNK.

 
Finally, MAPK phosphorylation, in response to various cytokines, shown by CBA to be present in CoMTB, was examined. CXCL-8, IL-6, IL-10, CCL-2, and CCL-5, as well as TNF-{alpha} and IL-1ß, were given singly or in combination at the concentrations found in CoMTB (Fig. 6D 6E 6F) . IL-1 induced phosphorylation of all three MAPKs, although not to the levels induced by CoMTB. None of the other factors tested, including TNF-{alpha}, induced MAPK phosphorylation in astrocytes. A combination of all tested cytokines and chemokines used at CoMTB-equivalent levels induced no additional MAPK phosphorylation than IL-1 acting alone.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we show that a monocyte-dependent network drives astrocyte MMP-9 secretion in response to M. tuberculosis infection. IL-1ß and TNF-{alpha} are known to be critical factors in the host response to pulmonary TB [46 47 48 ]. These cytokines are necessary but not sufficient for astrocyte MMP-9 secretion in response to M. tuberculosis. Intracellularly, CoMTB activates the p38, Erk, and Jnk MAPKs, which signal through the c-Jun, FosB, Fra-1, Fra-2, and JunB subunits of AP-1. Here, we demonstrate that IL-1ß but not TNF-{alpha} stimulates MMP-9 secretion from astrocytes through the MAPK pathways. Astrocyte-derived MMP-9 may contribute to the development of a tissue-destructive phenotype in the CNS.

We show that MMP-9 secretion is induced by conditioned media from monocytes infected with M. tuberculosis. This is consistent with our previous data, which show elevated CSF MMP-9 concentrations in patients within CNS-TB [6 ] and implicate astrocytes as a potential cellular source of MMP-9 in CNS-TB. Neither direct infection with M. tuberculosis nor stimulation with CoTB induced astrocyte MMP-9 secretion; therefore, the MMP-9-inducing activity of CoMTB is monocyte- rather than M. tuberculosis-derived. Astrocytes are by far the most populous CNS cell population and are distributed ubiquitously throughout the brain. As a 1:20 dilution of CoMTB was able to stimulate increased MMP-9 secretion, the active factors secreted by monocytes might diffuse through large regions of the CNS at levels high enough to activate astrocytes. Such MMP-9 secretion might play a role in the widespread tissue destruction induced in CNS-TB.

Increased MMP-9 secretion is induced by proinflammatory cytokines in a range of CNS diseases characterized by tissue-destructive pathology [49 ]. We established that CoMTB contains increased concentrations of the cytokines IL-1ß, IL-6, and TNF-{alpha} and the chemokines CCL-2, CCL-5, and CXCL-8 compared with CoMCon [18 ] (data not shown). By blocking TNF-{alpha} and IL-1ß activity, we show that these cytokines are important in the MMP-9-inducing effect of CoMTB. IL-1ß and TNF-{alpha} are present at low levels in healthy brain tissue, where an anti-inflammatory environment is maintained [50 , 51 ]. Current data are consistent with CNS studies showing increased MMP-9 secretion in response to IL-1ß and TNF-{alpha} in primary rat astrocytes [28 , 29 ]. However, even when these cytokines were blocked simultaneously, inhibition of MMP-9 secretion was incomplete. In addition, although high concentrations of TNF-{alpha} and IL-1ß induce MMP-9 secretion from astrocytes, physiological levels of these cytokines found in CoMTB only cause low-level MMP-9 secretion. These data demonstrate that other mediators derived from monocytes act with IL-1ß and TNF-{alpha} to increase MMP-9 secretion from astrocytes in response to CoMTB. These data contrast with our previous findings investigating monocyte-monocyte networks in the context of MMP-9 secretion in TB, where TNF-{alpha} but not IL-1 was found to be important [52 ]. Thus, control of MMP-9 secretion appears cell-specific. Our data suggest that IL-1 is the key regulator and support the hypothesis that differing responses within the modified immune system of the CNS may be responsible for the extensive tissue damage caused by CNS-TB. Similarly, IL-1 has a major role in the development of neurodegeneration and neuroinflammation in multiple sclerosis and CNS infections [53 , 54 ].

Chemokines play a critical role in the MMP response of monocytes, T cells, and pulmonary epithelial cells [18 , 40 , 52 ]. However, stimulation of astrocytes with cholera toxin, which activates the G-protein-linked pathways alone or in combination with TNF-{alpha} and IL-1ß, had no effect on astrocyte MMP-9 secretion. Similarly, PTX-induced inhibition of signaling through the G-protein-linked pathways had no effect on CoMTB-mediated MMP-9 secretion. In networks involving pulmonary epithelial cells, an M. tuberculosis-derived factor synergizes with TNF-{alpha} to induce MMP-9 secretion (work in progress). No such interaction with M. tuberculosis-derived factors occurs in astrocytes.

To understand further the astrocyte response to monocyte-derived cytokines, the activation of AP-1 was investigated. The strong transactivators FosB and c-Fos and the weaker transactivator JunB were shown to be most up-regulated by CoMTB. The weak transactivators Fra-1 and Fra-2 and stronger transactivator c-Jun were up-regulated to a lesser extent. This suggests that the overall effect of CoMTB on AP-1 is stimulatory. Future work will examine this further through detailed analysis of the MMP-9 promoter.

MAPKs are known to be involved in the activation of AP-1 DNA-binding activity [36 , 37 ]. We show that Erk, p38, and Jnk regulate astrocyte MMP-9 secretion in response to CoMTB. Depending on the stimuli and cell type, different MAPK activation profiles are required for MMP-9 up-regulation [55 , 56 ]. In a response similar to that observed in this investigation, TNF-{alpha}-dependent MMP-9 up-regulation in cortical rat astrocytes requires Erk, p38, and Jnk activation [55 ]. In contrast, TNF-{alpha}-dependent MMP-9 secretion from THP-1 monocytic cells required Erk activity only. Jnk and p38 inhibition was found to increase secretion of MMP-9 [57 ]. We determined that IL-1ß activated astrocyte MMP-9 secretion through the MAPK signaling pathways but that neither TNF-{alpha} nor a range of other cytokines and chemokines did so at pathophysiological concentrations. The degree of phosphorylation activated by IL-1ß is significantly lower than observed in CoMTB-stimulated cells. Thus, other mediators present in CoMTB interact with IL-1 to up-regulate the MAPK signaling cascade. Previous studies have shown that synergistic interactions between OSM and IL-1 up-regulate MMP-1 secretion [58 ], but although CoMTB contains OSM, no similar interaction was found in this model. This investigation is consistent with studies showing that in cultured rat astrocytes, MMP-9 secretion may be up-regulated by IL-1ß via the MAPK signaling pathways [28 , 32 ]. This is the first study to show that the monocyte-astrocyte network’s response to M. tuberculosis is dependent on the MAPK cascade to initiate MMP-9 secretion.

In summary, monocyte-dependent networks induce MMP-9 secretion from astrocytes in response to M. tuberculosis. IL-1 and TNF-{alpha} are essential but not sufficient for this MMP-9 induction. The MAPK signaling cascade plays a key role in IL-1-mediated MMP-9 secretion. TNF-{alpha} is using alternative pathways. Recently, MAPK signaling pathways have been identified as targets for a range of anti-inflammatory pharmaceuticals [59 , 60 ], as have therapeutics targeting IL-1-mediated inflammation [54 ]. The data presented in this paper suggest that such molecules may have the potential to restrict MMP-9 secretion, potentially reducing morbidity associated with CNS-TB.


   ACKNOWLEDGEMENTS
 
J. E. H. and J. A. G. were supported by a Ph.D. studentship and clinical training fellowship, respectively, from the Medical Research Council (UK). P. T. E. is supported by a UK Department of Health fellowship.

Received August 10, 2006; revised September 19, 2006; accepted October 5, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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