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(The FASEB Journal. 2007;21:356-365.)
© 2007 FASEB

IFN{gamma} synergizes with IL-1ß to up-regulate MMP-9 secretion in a cellular model of central nervous system tuberculosis

James E. Harris*, Marc Fernandez-Vilaseca*, Paul T. G. Elkington*, Donna E. Horncastle{dagger}, Manuel B. Graeber{ddagger} and Jon S. Friedland*,1

* Department of Infectious Diseases and Immunity and

{dagger} Department of Histopathology, Imperial College, Hammersmith Campus, London, UK;

{ddagger} Department of Neuropathology, Imperial College, Charing Cross Campus, London, UK

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Matrix metalloproteinase-9 (MMP-9) activity is implicated in pathogenesis of central nervous system tuberculosis (CNS-TB). IFN{gamma}, a key cytokine in TB, usually inhibits MMP-9 secretion. Addition of IFN{gamma} to conditioned media from M. tb-infected monocytes (CoMTB) resulted in a 7-fold increase in MMP-9 activity detected by gelatin zymography (P<0.01). In contrast, tissue inhibitor of metalloproteinase (TIMP)-1 and -2 secretion, measured by ELISA, was suppressed. Dexamethasone abolished the synergistic increase in MMP-9 activity. Interleukin (IL)-1ß in CoMTB is a critical mediator of synergy with IFN{gamma}, and IL-1ß alone synergizes with IFN{gamma} to increase MMP-9 secretion from 51 ± 31 to 762 ± 136 U. IL-1ß activity is dependent on p38 mitogen-activated protein (MAPK) kinase, which was found to be phosphorylated in tissue specimens from patients with CNS-TB. Extracellular signal regulated kinase (Erk) and p38 MAPK activation did not affect IFN{gamma} signaling pathways. Inhibition of janus-activated kinase (JAK)-2 by 50 µM AG540 decreased MMP-9 secretion to 124 ± 11.1 from 651 ± 229 U of activity (P<0.01). However, signal transducer and activator of transcription (STAT)-3 but not STAT-1 phosphorylation was synergistically up-regulated by IFN{gamma} and CoMTB. In summary, synergy between IL-1ß and STAT-3 dependent IFN{gamma} signaling is key in control of up-regulation of MMP-9 activity in CNS-TB and may be a significant mechanism of brain tissue destruction.—Harris, J. E., Fernandez-Vilaseca, M., Elkington, P. T. G., Horncastle, D. E., Graeber, M. B., and Friedland, J. S. IFN{gamma} synergizes with IL-1ß to up-regulate MMP-9 secretion in a cellular model of central nervous system tuberculosis.


Key Words: astrocyte • Mycobacterium tuberculosis • monocyte • gelatinase B • MAP kinase • JAK-STAT


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CENTRAL NERVOUS SYSTEM TUBERCULOSIS (CNS-TB) is the most dangerous form of tuberculous disease, which kills or causes clinically significant morbidity in half of those affected (1) . Damage to CNS tissues, including degradation of the blood-brain barrier, is a major cause of such morbidity and mortality in CNS-TB (2 3 4) . The mechanisms resulting in tissue destruction in CNS-TB are currently unclear. The matrix metalloproteinases (MMP) are a family of zinc-dependent proteases that are implicated in tissue-destructive pathology in pulmonary tuberculosis (5 , 6) . MMP activity is tightly regulated at the level of transcription and is controlled after secretion by tissue inhibitors of metalloproteinases (TIMP) (7) . MMP-9 (92 kDa collagenase) can degrade many structural components of the blood-brain barrier and CNS tissue matrix, including type IV collagen, laminins, and fibronectin (8 , 9) . Evidence that MMP-9 can damage CNS tissues is found in MMP-9 knockout mice, where blood-brain barrier disruption following ischemic injury is reduced compared with controls (10) . MMP-9 concentrations are increased in cerebrospinal fluid (CSF) samples from patients with CNS-TB (11) . We observed that MMP-9 concentrations were greater in tuberculosis compared with bacterial or viral meningitis and were associated with signs of local tissue CNS destruction and death (12) .

Astrocytes are the most numerous cell-type within the CNS and are integral to both innate immunity within the CNS and maintenance of the extracellular matrix (ECM) (13) . In normal physiology MMP-9 secretion is limited, and under these conditions astrocyte-derived MMP-9 allows tissue remodeling and neurite extension (14 , 15) . The role played by astrocytes in inflammatory tissue destruction in CNS-TB is unknown. However, increased astrocyte MMP-9 secretion is induced by proinflammatory stimuli, including IL-1ß (16) . During CNS-TB monocytes migrate to the CNS in large numbers (18) and, when stimulated with M. tuberculosis, increase secretion of IL-1ß (5 , 17) . MMP-9 secretion from IL-1-stimulated rat astrocytes is critically dependant on the mitogen-activated protein kinases (MAPK) p38, Erk, and Jnk (19) . In CNS-TB, the CSF concentrations of a range of proinflammatory cytokines including IL-1ß and IFN{gamma} are increased (20 21 22) . Both the extent of tissue destruction and CSF concentrations of IFN{gamma} and IL-1ß are higher in CNS-TB than in bacterial meningitis (22 23 24) .

IFN{gamma} is a critical component of the host response to M. tuberculosis. Mutations of the gene for the IFN-{gamma}-receptor high-affinity receptor (IFN-{gamma}R1) result in increased susceptibility to mycobacterial infection (25) . IFN{gamma} knockout mice develop a rapidly fatal infection characterized by increased tissue destruction when infected by M. tuberculosis (15) . Large numbers of T-cells enter the CNS during CNS-TB (26) and are largely responsible for increased CSF IFN{gamma} concentrations detectable in patients (23) . IFN{gamma} signaling has been investigated in detail. In brief, IFN{gamma} binds transmembrane receptors on the target cell. Binding of IFN{gamma} induces receptor tyrosine phosphorylation by JAK-1 and -2, resulting in recruitment of signal transducer and activator of transcription-1 (STAT-1) homodimers. STAT-1 is phosphorylated and then translocates to the nucleus where it binds directly to specific sites in the target gene’s promoter (27) . An important additional pathway activated by IFN{gamma} results in phosphorylation of STAT-3 (28) . The mechanism resulting in this activation is currently unclear but may be independent of the IFN{gamma} receptor and appears to be cell-type specific (29) . In addition, IFN{gamma} may stimulate transcriptional activity through activation of MAPKs (30 , 31) .

Previous data suggest that the interferons tend to inhibit MMP-9 secretion (3235), although studies have shown borderline up-regulation of MMP-9 by IFN{gamma} in bladder cells (36) . IFN{gamma} inhibits TNF-{alpha}-induced MMP-9 secretion in a range of human cell types (37) . In the CNS, IFN{gamma} inhibited MMP-9 expression by PMA-stimulated human astroglioma cell lines and primary astrocytes through a STAT-1 dependant mechanism (38) . The interaction between astrocytes and IFN{gamma} in CNS-TB has not been investigated. We have investigated the hypothesis that IFN{gamma} acts as a modulator of monocyte-astrocyte cytokine networks in CNS-TB.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
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, USA). Triton X-100 was purchased from VWR (Poole, UK). Coomassie blue tablets were obtained from Pharmacia Biotech (Uppsala, Sweden). For Western blotting, rabbit antibodies against phosphorylated and nonphosphorylated forms of human c-Jun NH2-terminal kinase (JNK), ERK, p38, STAT-1, STAT-2, STAT-3, STAT-5, and STAT-6, as well as peroxidase-conjugated anti-Rabbit IgG, were purchased from Cell Signaling Technology (Beverly, MA, USA). The JNK, ERK, and p38 inhibitors SP600125, PD98059, and SB2030580 and the JAK-2 inhibitor AG490 were purchased from Merck Biosciences (Nottingham, UK). For immunohistochemistry, mouse monoclonal anti-human phosphorylated-p38Ab (clone M8177 and Polyclonal Rabbit anti-human GFAP antibody (Ab) were purchased from DakoCytomation (Ely, UK). All other reagents were purchased from Sigma-Aldrich (Poole, UK).

M. tuberculosis culture
M. tuberculosis H37-Rv was maintained in Middlebrook 7H9 medium supplemented with 10% ADC enrichment medium, 0.2% glycerol, 0.02% Tween-80, and 2.5 µg/ml amphotericin with agitation. M. tuberculosis was used at midlog growth phase at optical density (OD) 0.60 (Biowave Cell Density Meter, WPA, Cambridge, UK) in all experiments, corresponding to 1 x 108 CFU/ ml. M. tuberculosis endotoxin level was measured by the amoebocyte lysate (Associates of Cape Cod, East Falmouth, MA, USA).

Cell culture
Human astrocyte cell line U373-MG (ECACC No. 89081403) was maintained in MEME supplemented with 10% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, 1% nonessential amino-acids, and 100 µg/ml ampicillin, according to suppliers’ 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, Little Chalfont, UK) followed by adhesion purification. Monocyte purity was more than 95% by FACS analysis. Monocytes were plated out in RPMI with 2 mM glutamine and 10 µg/ml ampicillin and infected with M. tuberculosis. Conditioned medium was harvested at 24 h followed by filtration through a 0.2 µm Anopore membrane to remove M. tuberculosis (39) . Conditioned media from infected monocytes were termed CoMTB, media from uninfected monocytes were conditioned media from control monocytes (CoMCon).

Once confluent, U373-MG cells were stimulated with a 1:5 dilution of CoMTB or CoMCon in MEME unless otherwise stated. Supernatant was harvested from astrocytes at 72 h and spun at 12,000 RCF for 5 min to remove cellular debris and frozen immediately. ELISA analysis showed that CoMTB did not contain IFN{gamma} (data not shown).

Zymography
Standard methodology for gelatin zymography was used to detect MMP-9 activity in samples (40) . In brief, standards and prepared cell-supernatants were loaded with 5x loading buffer (0.25M Tris, pH 6.8; 50% glycerol; 5% SDS; bromphenol blue) and run on 11% acrylamide gels impregnated with 0.1% gelatin as substrate. After ~3.5 h at 180V (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 2 ng recombinant MMP-9 (Calbiochem, Nottingham, UK) to standardize between gels. Gel images were photographed with a Transilluminator (UVP) followed by proteolytic band quantification using LabWorks (version 4.5). The digitized results of each sample were normalized to the standard readings. For calcium chelation to inhibit MMP activity, 10 mM EDTA was added to the collagenase buffer.

ELISAs
Sandwich ELISAs were used to measure secretion of IFN{gamma} and tissue inhibitor of metalloproteinase (TIMP)-1 and -2 according to manufacturers instructions (R&D Systems, Minneapolis, MN, USA). For TIMP-1 and -2 the lower limit of detection was 31 pg/ml, for IFN{gamma} the lower limit of detection was 16 pg/ml.

Western blot analysis for detection of MAPK and STAT phosphorylation
To investigate phosphorylation of JNK, p38, ERK, STAT-1, and STAT-3, confluent cells were stimulated with conditioned media and incubated until a specific time point. Cells were then washed with ice-cold PBS and then scraped into ice-cold SDS sample buffer (62.5 mM Tris/2% SDS/10% glycerol/50 mM DL-dithiothreitol/0.01% bromphenol blue). Samples were frozen until required. Each sample (40 µl quantities) was heat-denatured and run on a 10% acrylamide gel at 200V (Running buffer 25 mM Tris base, 192 mM glycine, 0.1% SDS) for 3 h. After separation, proteins were transferred to a nitrocellulose membrane (GE, Little Chalfont, UK) and blocked for 1 h with 5% milk protein/0.1% Tween-20. Then membranes were incubated with the primary antibodies (1:1000 dilution) overnight at 4°C. After washing, the membrane was incubated with peroxidase-conjugate secondary Ab (1:2000 dilution), for 1 h. Protein bands were visualized on Hyperfilm enhanced chemiluminescence (ECL) (GE, Little Chalfont, UK) by chemiluminescence.

Immunohistochemistry
To examine the spatial distribution of phosphorylated-p38 in infected and uninfected CNS tissue in vivo, immunohistochemistry for phophoryla ted-p38 and glial fibrilliary acid protein (GFAP) was performed on biopsies from five patients with culture-proven M. tuberculosis infection and a noninfectedcontrol. Sections (4 µm) were dewaxed, and endogenous peroxidase activity was blocked with 0.6% hydrogen peroxide for 15 min. Sections were microwaved for 20 min in citrate buffer (0.01 M citrate, pH 6.0) and blocked with 5% normal goat serum for 10 min. The primary Abs (phosphorylated p38 at 1:200 and GFAP at 1:500 dilution) were applied in 0.01 M PBS/azide/BSA for 1 h at room temperature. Ab was detected with the Menarini nonbiotinylated kit according to the manufacturer’s instructions. Peroxidase activity was developed with the 3,3'-diaminobenzidine system (Menarini, Florence, Italy). Slides were counterstained with Cole’s hematoxylin, dehydrated, and mounted.

Data presentation and statistical analysis
Data are presented as mean ± SD of three samples and represent at least two experiments performed in triplicate unless otherwise stated. Statistical analysis was performed using statistical Packages for the Social Sciences (SPSS). 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. In all graphs, * represents a P value <0.05; ** represents a P value <0.01.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
IFN{gamma} synergizes with CoMTB to up-regulate MMP-9 but not TIMP-1/2 secretion
We examined the effect of CoMTB and IFN{gamma} alone and in combination on MMP-9 secretion from astrocytes stimulated with conditioned media from M. tuberculosis-infected monocytes (CoMTB). A 1:5 dilution CoMTB: MEME induced 215 ± 16 U MMP-9 secretion (Fig. 1 A; P<0.05). IFN{gamma} alone had no effect. Unexpectedly, IFN{gamma} at 50 ng/ml synergized with CoMTB, resulting in a 7-fold increase in MMP-9 secretion compared with CoMTB alone (P<0.05). In isolation, 50 ng/ml IFN{gamma} did not induce MMP-9 secretion from astrocytes. The synergy between IFN{gamma} and CoMTB was dose-dependent and significant even with 5 ng/ml IFN{gamma} (P<0.05). IFN{gamma} enhances MMP-9 secretion from CoMTB stimulated astrocytes in both the U373-MG and U87-MG astrocytes cell lines (data not shown). IFN{gamma} did not increase MMP-9 secretion from fibroblasts or a human microglial cell line stimulated with CoMTB (data not shown).


Figure 1
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  		Figure 1. Stimulation of astrocytes with CoMTB and IFN{gamma} upregulates MMP-9 but not TIMP-1 and -2 secretion. Cell culture media were collected at 72 h. A) Representative zymogram showing astrocyte MMP-9 secretion up-regulated synergistically by CoMTB and increasing doses of IFN{gamma}. B) Graph is the densitometric analysis of gelatin zymography showing that CoMTB and IFN{gamma} synergize to up-regulate MMP-9 secretion, but that IFN{gamma} alone has no effect. C) TIMP-1 secretion following stimulation with CoMTB, IFN{gamma}, or a combination of stimuli. D) TIMP-2 secretion following stimulation with CoMTB, IFN{gamma}, or a combination of stimuli. All data are representative of two independent experiments performed in triplicate.

In contrast, IFN{gamma} inhibited CoMTB-stimulated astrocyte secretion of TIMP-1 and TIMP-2 compared with CoMTB alone. TIMP-1 secretion from CoMTB-stimulated astrocytes was inhibited by 31.1 ± 10.6% (P<0.05) and 40.9 ± 13.1% (P<0.01) by 5 ng/ml and 50 ng/ml IFN{gamma}, respectively (Fig. 1B ). TIMP-2 secretion decreased 40.5 ± 28.5% in astrocytes stimulated with CoMTB and 50 ng/ml IFN{gamma} compared with CoMTB alone (Fig. 1C ; P<0.05). In the absence of CoMTB, neither TIMP-1 nor TIMP-2 secretion was significantly affected by IFN{gamma}.

Dexamethasone antagonizes IFN{gamma} and CoMTB-stimulated astrocyte MMP-9 secretion
The corticosteroid dexamethasone is effective at reducing mortality in patients with CNS-TB through mechanisms not yet defined (41) . Incubation of IFN{gamma} and CoMTB-stimulated astrocytes with 0.1 µM dexamethasone results in a 75.2 ± 4.7% decrease in MMP-9 secretion (Fig. 2 ; P<0.01). When CoMTB and IFN{gamma} stimulated astrocytes are coincubated with 10 mM of dexamethasone, MMP-9 secretion decreases by 86%, from 2423.7 ± 195.1 to 339.1 ± 90.5 U (P<0.01). Dexamethasone (10 mM) also inhibits MMP-9 secretion from astrocytes stimulated with CoMTB alone from 507.5 ± 62.7 to 262.4 ± 41.5 U (P<0.01).


Figure 2
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  		Figure 2. Dexamethasone suppresses MMP-9 secretion from astrocytes costimulated with CoMTB and IFN{gamma}. Data represent two independent experiments performed in triplicate. Graph represents densitometric analysis of gelatin zymography. *Represents a P-value <0.05. **Represents a P-value <0.01.

IL-1ß in CoMTB synergizes with IFN{gamma} to up-regulate astrocyte MMP-9 secretion
We next examined the role of IL-1, a key cytokine in TB, in mediating synergy between CoMTB and IFN{gamma}. Inhibition of IL-1 signaling with IL-1Ra inhibited synergy between CoMTB and IFN{gamma} by 54 ± 11% (Fig. 3 A; P<0.01). However, this MMP-9 concentration was significantly higher than that from cells stimulated with CoMTB alone (P<0.05).


Figure 3
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  		Figure 3. IL-1ß in CoMTB synergizes with IFN{gamma} to induce up-regulation of astrocyte MMP-9 secretion. Graphs are densitometric analysis of gelatin zymography, where the y-axis represents MMP-9 activity (units). A) IL-1Ra inhibits MMP-9 secretion from astrocytes stimulated with both CoMTB and IFN{gamma} (50 ng/ml). B) IFN{gamma} but not TNF{alpha} synergizes with IL-1ß to induce astrocyte MMP-9 secretion. C) The effect of IFN{gamma} on MMP-9 secretion is concentration-dependent. All data are representative of two independent experiments performed in triplicate. *P value <0.05, **P value <0.01.

Next we examined whether astrocyte MMP-9 secretion is affected by synergy between IFN{gamma} and IL-1ß in the absence of additional factors in CoMTB. IL1ß (5 ng/ml) synergizes with 25 ng/ml IFN{gamma} to cause MMP-9 secretion of 762.2 ± 136 U of activity (Fig. 3B ; P<0.01). In contrast, IFN{gamma} (25 ng/ml) and IL-1ß (5 ng/ml), when used alone as stimuli, do not drive MMP-9 secretion or 51 ± 30.7 U of MMP-9 activity, respectively. The synergy between IL-1ß and IFN{gamma} is dose-dependent. For example, when astrocytes are stimulated with 5 ng/ml IL-1ß and 5 ng/ml IFN{gamma}, MMP-9 secretion of 501.1 ± 69.4 U is observed (P<0.01, Fig. 3C ).

ERK activity is required for synergy between IFN{gamma} and IL-1ß/CoMTB
In view of the involvement of MAPK in proinflammatory signaling pathways, we blocked Erk activity using the specific inhibitor PD98059 (42 , 43) . PD398059 (10 µM) supresses MMP-9 up-regulation in response to CoMTB and IFN{gamma} by 77.5 ± 12.6% (P<0.05; Fig. 4 A). MMP-9 secretion from astrocytes stimulated with CoMTB and IFN{gamma} in the presence of PD98059 was significantly lower than that in cells stimulated with CoMTB alone (P<0.01). No affect on cell viability was observed at these concentrations.


Figure 4
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  		Figure 4. ERK activity is required for synergy between IFN{gamma} and CoMTB. A) Graph represents densitometric analysis of gelatin zymography showing effects of 2.5, 10, and 30µM PD98059 on MMP-9 secretion from IFN{gamma} and CoMTB stimulated astrocytes. Data are from two independent experiments performed in triplicate. B) Western blots representative of three independent experiments showing Erk phosphorylation in response to IFN{gamma} and/ or CoMTB and IL-1ß.

Next we examined whether IFN{gamma} affects Erk phosphorylation in CoMTB-or IL-1ß-stimulated astrocytes. Erk-phosphorylation is constitutive and is only marginally up-regulated by CoMTB. No additional phosphorylation is induced by the addition of 1, 5, or 25 ng/ml IFN{gamma} (Fig. 4B ). When IFN{gamma} is applied to astrocytes in the absence of CoMTB, Erk-phosphorylation levels are equivalent to those seen in unstimulated astrocytes. IL-1ß induced a slight increase in Erk-phosphorylation. Thus, Erk activity is required for MMP-9 secretion, but up-regulation does explain the synergy between CoMTB and IFN{gamma}.

P38 MAPK is required for synergy between CoMTB and IFN{gamma}
Next, p38 activity was inhibited with the specific inhibitor SB203580 (42 , 44) . SB203580 (0.5 µM) inhibited MMP-9 secretion from astrocytes stimulated with CoMTB and 25 ng/ml IFN{gamma} by 89.1 ± 5.6% (Fig. 5 A; P<0.01). At this SB203580 concentration, MMP-9 secretion is not significantly different to that in astrocytes stimulated with CoMTB alone. In contrast to Erk, p38 phosphorylation is not constitutive in U373-MG astrocytes but is induced by CoMTB within 30 min (Fig. 5B ). Costimulation of CoMTB with IFN{gamma} at 1, 5, or 25 ng/ml resulted in no additional p38 phosphorylation. IFN{gamma} alone induced no p38 phosphorylation in astrocytes. IL-1ß in isolation caused p38 phosphorylation, which was not increased by costimulation with IFN{gamma}.


Figure 5
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  		Figure 5. p38 activity is required for synergy between IFN{gamma} and CoMTB and is activated in vivo. A) Graph is densitometric analysis of gelatin zymography showing effects of SB203580 on MMP-9 secretion from IFN{gamma} and CoMTB-stimulated astrocytes. Data represent two independent experiments performed in triplicate. B) Western blots representative of three independent experiments, showing p38 phosphorylation in response to IFN{gamma}, CoMTB, and IL-1ß alone or in combination. C) The distribution of phosphorylated-p38 in clinical samples from patients with CNS-TB. Expression of phosphorylated-p38 is up-regulated in enlarged reactive astrocytes that are found in association with tuberculous brain tissue. These cells show greater nuclear localization of phosphorylated-p38 in CNS-TB brain tissue compared with controls. Immunohistochemistry for GFAP using adjacent tissue sections demonstrates a very significant up-regulation of GFAP expression and marks numerous astrocyte processes confirming the presence of reactive astrocytosis. For patient details see text. The arrows indicate reactive astrocytes expressing GFAP or phosphorylated-p38.

To investigate the clinco-pathological significance of these findings, the distribution of phosphorylated-p38 in clinical samples from five patients with CNS-TB was examined (Fig. 5C ). Expression of phosphorylated-p38 is up-regulated in astrocytes associated with tuberculous tissue compared with control brain tissue. Phosphorylated p38 also shows greater nuclear localization in astrocytes and neuronal cells in CNS-TB brain tissue compared with controls. GFAP expression confirmed the presence of activated astrocytes in this tissue.

The role of the JAK/STAT pathway in synergistic up-regulation of astrocyte MMP-9 secretion
Since the point of synergy was not the p38 or Erk MAPK signaling pathways, we investigated the JAK/STAT pathway. JAK-2 activity was blocked with 10 µM AG490, which significantly inhibits MMP-9 secretion from astrocytes stimulated with CoMTB and IFN{gamma} (Fig. 6 A; P<0.05). This effect was dose-dependent, and 50 mM AG490 decreased MMP-9 secretion from astrocytes stimulated with CoMTB and IFN{gamma} to a level not significantly different to that found in astrocytes stimulated with CoMTB alone. The observed synergy was abolished at this concentration (P<0.01). However, 50 mM AG490 did not inhibit MMP-9 secretion from astrocytes stimulated with CoMTB alone. No effect on cell viability was observed when AG490 was used at these concentrations.


Figure 6
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  		Figure 6. JAK/STAT activity is required for synergy between IFN{gamma} and CoMTB. A) Graph is densitometric analysis of gelatin zymography showing effects of 0.01, 10, and 50 µM AG490 on MMP-9 secretion from IFN{gamma} and CoMTB stimulated astrocytes. Data represent two independent experiments performed in triplicate. B) Western blots representative of three independent experiments, showing phosphorylation of STAT-1 (top) and STAT-3 (bottom) in response to IFN{gamma}, CoMTB, and IL-1ß alone or in combination.

JAK-2 is key in STAT-1 and -3 dependent signaling, and therefore phosphorylation of STAT-1 and STAT-3 was examined (Fig. 6B ). IFN{gamma} induces STAT-1 phosphorylation in a dose-dependent manner. However, no difference was found in STAT-1 phosphorylation levels between cells stimulated with either IFN{gamma} (50 ng/ml) alone or IFN{gamma} (50 ng/ml) in the presence of CoMTB or IL-1ß. Neither IL-1ß nor CoMTB activated STAT-1 phosphorylation.

Phosphorylation of STAT-3 at tyrosine 705 is activated by stimulation of astrocytes with CoMTB alone. IFN{gamma} causes low-level phosphorylation STAT-3 at tyrosine 705. Costimulation with CoMTB and IFN{gamma} results in synergistically higher phosphorylation than with either CoMTB or IFN{gamma} alone. IL-1ß does not phosphorylate STAT-3 at this site, and costimulation of astrocytes with both IL-1ß and IFN{gamma} results in no additional phosphorylation above that seen with IFN{gamma} alone. STAT-3 also contains a phosphorylation site at serine 727, which is constitutively phosphorylated in U373-MG cells. Some additional phosphorylation of serine 727 was induced by IL-1ß but not IFN{gamma}. No synergy was apparent between CoMTB and IFN{gamma}.

To examine whether crosstalk may be occurring between p38 and ERK and STAT-1 and STAT-3, we performed a series of blocking experiments (Fig. 7 ). We show that inhibition of ERK and p38 with PD98059 and SB203580, respectively, had no effect on CoMTB-induced phosphorylation of the STAT-3 tyrosine 705 site. STAT-1 phosphorylation in response to IFN{gamma} was also unaffected by inhibition of ERK and p38. Therefore, these pathways operate independently in this model.


Figure 7
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  		Figure 7. Absence of crosstalk occurs between the JAK/STAT and MAPK pathways in response to costimulation with CoMTB and IFN{gamma}. Western blots representative of two independent experiments, showing the effect of PD98059 (30 µM) and SB203580 (10 µM) on the phosphorylation of STAT-1 (top) and STAT-3 (bottom).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Mycobacterium tuberculosis is extremely well adapted to infection of the lungs, and infection may persist in a state that facilitates transmission for many years without killing the host. In contrast, for both M. tuberculosis and its host, infection of the CNS is an evolutionary dead end. The bacilli that invade the brain cannot be transmitted to new hosts and without therapy, M. tuberculosis within the CNS will almost certainly result in death of the infected host (1) . Therefore, neither pathogen nor host can evolve survival mechanisms for this scenario. In contrast to the lungs, growing evidence suggests that immune reactions in the brain lead instead to inflammation, MMP secretion, and tissue destruction in the unique immunological setting of the CNS. This investigation supports this hypothesis and implicates the astrocyte and IFN{gamma} as key amplifier of inflammatory tissue destruction in CNS-TB.

The key finding of the present study is that IFN{gamma} and CoMTB synergize to induce high-level MMP-9 secretion from human astrocytes. CoMTB alone induces significant MMP-9 secretion, but, in combination with IFN{gamma}, secretion increases by a factor of 7. In contrast, IFN{gamma} alone does not affect MMP-9 secretion. Previously, IFN{gamma}’s effect on MMP-9 secretion from a range of cell types has been shown to be primarily inhibitory. For example, PMA and TNF-{alpha}-induced MMP-9 secretion from astrocytes and astrocytoma-cells was inhibited by IFN{gamma} (45) . TNF-{alpha}-induced MMP-9 secretion from monocytes is inhibited by IFN{gamma} through STAT-1-dependent sequestration of the coactivators CBP/p300 (38 , 46) . To our knowledge this study is the first showing IFN{gamma} increasing the expression of MMP-9 through synergy with other mediators. The effects of this increase in secretion on MMP-9 activity in vivo could be amplified by the fact that IFN{gamma} up-regulates a range of genes that activate pro-MMP-9, including the urokinase-type plasminogen activator and its receptor (47) .

Secretion of the specific MMP-9 inhibitor TIMP-1 is somewhat decreased by IFN{gamma}, either alone or in combination with CoMTB. This is consistent with the increased CSF concentrations of MMP-9 but not TIMP-1, which we found in patients with CNS-TB, which were associated with increased morbidity and mortality (12) . Together, the data suggest that the MMP-9/TIMP-1 balance is pushed toward facilitating tissue damage in CNS-TB and that IFN{gamma} is an amplifier of this effect.

Persistently high CSF concentrations of IFN{gamma} are an established feature of CNS-TB (22 , 23 , 26) . However, no data linking IFN{gamma} directly to MMP-9 secretion in CNS-TB exist. IFN{gamma} concentrations used in our experiments are similar to those seen in vivo during CNS-TB. CSF IFN{gamma} concentrations of 0.75 ng/ml have been reported (23 , 26) , which are slightly lower that the significant effects documented with 5 ng/ml recombinant IFN{gamma}. It is probable that in vivo the IFN{gamma} concentration that astrocytes encounter in the localized environment around foci of infection is higher than that found diluted in the CSF. IFN{gamma} levels are particularly increased in tuberculous as opposed to bacterial meningitis as a result of a fundamentally different immune response in CNS-TB (22 23 24 , 26 , 48) . Our data indicate that such high levels of IFN{gamma} found in tuberculous meningitis might be important in driving the elevated MMP-9 concentrations, which we documented in tuberculous but not in other forms of meningitis that were correlated with morbidity and mortality (12) .

In the periphery IFN{gamma} is secreted by T-cells at high concentrations and is responsible for helping to co-ordinate innate and adaptive responses to M. tuberculosis. During CNS-TB, cells from the peripheral immune system enter the CNS in large numbers (18 , 49) . Our data indicate that cytokines released by infiltrating monocytes from the peripheral innate immune system interact with IFN{gamma} from infiltrating T-cells to increase the tissue destructive potential of the CNS by activating astrocytes. In vivo it is likely that other CNS resident cells will be involved in this process, most notably the microglial immune network (50) . The cytokine secretion profile of microglia in response to M. tuberculosis is likely to be similar to that from monocytes (51) and microglia may also play a role in the activation of MMP-9 secretion from astrocytes.

Dexamethasone is used as an adjunct to chemotherapy in the treatment of tuberculous meningitis, where it reduces morbidity (41) . Here we show that dexamethasone can inhibit the synergistic up-regulation of MMP-9 by IFN{gamma} and CoMTB. Recently, Simmonds et al. have shown that out of a range of immunological and routine biochemical indices, only IFN{gamma} and CSF total protein content were shown to be significantly reduced by dexamethasone (26) . MMP-9 concentrations were not examined in this study, but these findings together with our data suggests that dexamethasone might target astrocyte MMP-9 secretion both directly and by inhibiting IFN{gamma}-dependent MMP-9 release.

In the present study, we found that IL-1ß in CoMTB is involved in the synergy between CoMTB and IFN{gamma}. In addition, we show that IFN{gamma} and IL-1ß synergize to increase MMP-9 secretion in the absence of other factors. Such MMP-9 secretion is less than in astrocytes stimulated with IFN{gamma} and CoMTB. Although IL-1ß may synergize with oncostatin M to induce MMP-1, -3, -8, -13, and -14 (52 , 53) this is the first study to show synergy between IL-1ß and IFN{gamma}-inducing MMP-9 secretion. Interestingly, CSF concentrations of IL-1ß did not differ significantly between tuberculous meningitis and other forms of bacterial meningitis (54 , 55) , but significant differences exist in CSF IFN{gamma} concentrations between tuberculous and other forms of meningitis (22 , 23 , 26) . This finding supports the hypothesis that IFN{gamma} plays the key role in the development MMP-9-induced tissue destruction in CNS-TB. The finding that IFN{gamma} and IL-1ß synergize to up-regulate astrocyte MMP-9 secretion has implications in diseases such as Alzheimer’s, where MMP-9 has been implicated in the development of pathology and both IL-1ß and IFN{gamma} are over-expressed (56 , 57) .

IL-1ß-induced MMP-9 secretion from astrocytes has been shown to be dependent on Erk and p38 activity (19) . Here we show that synergistic up-regulation of MMP-9 secretion by IFN{gamma} and CoMTB is dependent on the activity of p38 and Erk. Increased nuclear localization of activated p38 was also observed in astrocytes in vivo, in tissue from patients with CNS-TB. The molecular mechanisms controlling nuclear translocation of p38 are not fully understood, with evidence suggesting that activation of p38 can proceed or follow translocation to the nucleus (58 , 59) . Costimulation with CoMTB and IFN{gamma} did not increase p38 phosphorylation. IFN{gamma} does not affect these MAP kinase signaling pathways directly, through activation of secondary mediators, or by crosstalk from the JAK-STAT pathway. Although IL-1ß phosphorylates both p38 and Erk, IFN{gamma} induced no additional activation in costimulation experiments. Thus, although Erk and p38 are important mediators of MMP-9 secretion, they do not drive synergy between CoMTB and IFN{gamma}.

A role for the JAK/ STAT signaling pathways in the IFN{gamma}- and CoMTB-dependent synergistic increase in MMP-9 concentrations was demonstrated by use of a specific JAK-2 inhibitor. JAK-2 is required for signaling via STAT-1 and STAT-3 (27) . STAT-3 is induced by both CoMTB and IFN{gamma} and, in combination, these stimuli induce synergistically greater tyrosine phosphorylation and thus activation of STAT-3. The MMP-9 promoter contains multiple putative binding sites for STAT-3, and mammary epithelial cells transformed with a constitutively dimerized form of STAT3 (STAT-3-C) have increased MMP-9 secretion (60) . Although constitutive tyrosine phosphorylation of STAT-3 is a common feature of many cell lines (60) , our data show that U373-MG astrocytes are not constitutively phosphorylated at tyrosine 705. STAT-3 is most commonly activated by molecules binding the gp130 receptor including oncostatin M and neurotrophic factors (61 , 62) . CoMTB induces tyrosine phosphorylation of STAT-3 by itself and contains several molecules that can bind gp130 such as IL-6. Kaur et al. show that continuous exposure of neuronal cells to gp130 family cytokines greatly enhances the STAT-3 response to IFN{gamma} (61) and such a mechanism might be operating here.

In contrast to STAT-3, the MMP-9 promoter does not contain binding sites for STAT-1 and IFN{gamma} alone does not drive MMP-9 secretion, so any effect that this pathway might have on MMP-9 secretion would be through activation of other genes. STAT activation of activating protein (AP)-1 subunit transcription has been suggested as a potential mechanism to explain STAT involvement in synergistic MMP-1 up-regulation by oncostatin M and IL-1 (52) . The MMP-9 promoter contains activator protein-1 (AP-1) binding sites, and STAT-1-dependent activation of AP-1 subunit transcription might lead to increased MMP-9 secretion. However, although IFN{gamma} activates STAT-1 phosphorylation in astrocytes, neither CoMTB nor IL-1ß had any effect on this condition, which indicates that finding is not a key path in the synergistic up-regulation of MMP-9 secretion. IFN{gamma} can activate MAPK phosphorylation through a STAT-1 dependent mechanism, which is still poorly defined (31 , 63 , 64) . However, inhibition of Erk and p38 pathways did not show evidence of crosstalk between the MAPK cascades and the STAT pathways in our model.

In summary, astrocyte MMP-9 secretion is synergistically up-regulated by CoMTB and IFN{gamma}. IL-1ß is important but not sufficient in induction of this synergy and can synergize with IFN{gamma} to induce MMP-9 in the absence of other monocyte-derived factors. The STAT-3 signaling pathway is activated synergistically by IFN{gamma} and CoMTB via a MAPK-independent mechanism. This finding may play a critical role in inducing MMP-9 secretion and tissue destruction in CNS-TB.


   ACKNOWLEDGMENTS
 
J. E. Harris was supported by a Medical Research Council (UK) Ph.D. studentship.

Received for publication August 10, 2006. Accepted for publication September 6, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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