Mycobacterium tuberculosis, but Not Vaccine BCG, Specifically Upregulates Matrix Metalloproteinase-1
http://www.100md.com
《美国呼吸和危急护理医学》
Department of Infectious Diseases and Department of Histopathology, Imperial College, Hammersmith Campus, London
School of Biological Sciences, University of East Anglia, Norwich, United Kingdom
ABSTRACT
Rationale: Pulmonary cavitation is fundamental to the global success of Mycobacterium tuberculosis. However, the mechanisms of this lung destruction are poorly understood. The biochemistry of lung matrix predicts matrix metalloproteinase (MMP) involvement in immunopathology.
Methods: We investigated gene expression of all MMPs, proteins with a disintegrin and metalloproteinase domain, and tissue inhibitors of metalloproteinases in M. tuberculosis–infected human macrophages by real-time polymerase chain reaction. MMP secretion was measured by zymography and Western analysis, and expression in patients with pulmonary tuberculosis was localized by immunohistochemistry.
Results: MMP-1 and MMP-7 gene expression and secretion are potently upregulated by M. tuberculosis, and no increase in tissue inhibitor of metalloproteinase expression occurs to oppose their activity. Dexamethasone completely suppresses MMP-1 but not MMP-7 gene expression and secretion. In patients with active tuberculosis, macrophages express MMP-1 and MMP-7 adjacent to areas of tissue destruction. MMP-1 but not MMP-7 expression and secretion are relatively M. tuberculosis specific, are not upregulated by tuberculosis- associated cytokines, and are prostaglandin dependent. In contrast, the vaccine M. bovis bacillus Calmette-Guerin (BCG) does not stimulate MMP-1 secretion from human macrophages, although M. tuberculosis and BCG do upregulate MMP-7 equally. BCG-infected macrophages secrete reduced prostaglandin E2 concentrations compared with M. tuberculosis–infected macrophages, and prostaglandin pathway supplementation augments MMP-1 secretion from BCG-infected cells.
Conclusions: M. tuberculosis specifically upregulates MMP-1 in a cellular model of human infection and in patients with tuberculosis. In contrast, vaccine BCG, which does not cause lung cavitation, does not upregulate prostaglandin E2–dependent MMP-1 secretion.
Key Words: macrophage matrix metalloproteinases Mycobacterium tuberculosis pathology prostaglandin E
Mycobacterium tuberculosis (MTb) infects approximately one-third of the world's population and kills 2 to 3 million people each year (1, 2). Pulmonary cavitation is vital to the transmission of MTb and favors bacterial persistence (3). The cavity is an immunoprivileged site, with MTb replicating freely in the cavity wall (3). After treatment of tuberculosis (Tb), opportunistic pathogens may colonize the residual cavity (4). The ability to cause pulmonary cavitation in the immunocompetent host differentiates MTb from the vaccine strain Mycobacterium bovis bacillus Calmette-Guerin (BCG). The standard animal model of Tb is the mouse, which is valuable in studying immunity to MTb (5), but the pathology of murine Tb is divergent from that of human Tb, being purely fibrotic without cavitation (5, 6). Guinea pigs develop granulomas similar to those in humans but rarely cavitate, whereas rabbits develop cavitation with BCG, which is not a pulmonary pathogen in humans (5). Therefore, mechanisms by which MTb causes tissue destruction are best investigated in primary human cells and by clinical studies.
Proteases from inflammatory cells have been hypothesized to cause tissue destruction in Tb since the era of Koch and Virchow (7). For cavitation to occur, the extracellular matrix of the lung must be degraded. Fibrillar type I collagen provides the tensile strength of the matrix and elastin allows distensibility (8). Type I collagen is highly resistant to enzymatic degradation and only matrix metalloproteinases (MMPs) are able to cleave it at neutral pH (9). Excess MMP activity in the lung causes tissue destruction. For example, mice constitutively overexpressing MMP-1 (interstitial collagenase) develop emphysema (10) and MMPs are implicated in other animal models of emphysema (11–13).
MMPs are a family of zinc-binding proteases that collectively degrade all components of the extracellular matrix (14) and are from the metalloprotease class that includes the family of proteins with a disintegrin and metalloproteinase domain (ADAMs). MMP-1, MMP-8, and MMP-13 are the primary enzymes responsible for degradation of type I collagen (15). Multiple MMPs can degrade elastin, including MMP-2, MMP-7, MMP-9, and MMP-12 (15). MMP-7 (matrilysin) is the most potent elastase produced by macrophages (16). In addition to matrix remodeling, MMPs have important roles in immunity, such as the proteolytic processing of chemokines and cytokines (17, 18). MMP activity is regulated by multiple mechanisms, including transcriptional control, secretion as proenzymes that require proteolytic activation, compartmentalization, and secretion of specific inhibitors (tissue inhibitors of metalloproteinases [TIMPs]) (17).
MMP expression changes with cellular differentiation: macrophages secrete a broader spectrum and greater quantities of these enzymes than do monocytes (19, 20). Macrophage MMP production is regulated primarily through a pathway dependent on prostaglandin E2 and cAMP (21–23). Macrophages are a principal source of MMPs at inflammatory loci (24). In Tb, macrophages form the central core of the granuloma and are the key effector of the host immune response to MTb (25). In pulmonary MTb infection, excessive proteolytic activity causes matrix breakdown, caseation of the granuloma, liquefaction, and finally cavitation (26). We hypothesize that MMPs are critical to this pulmonary destruction in Tb.
This work has been previously presented in part in the form of abstracts (27, 28).
METHODS
See the online supplement for additional details of the following methods.
MTb and BCG Culture
M. tuberculosis H37Rv and M. bovis BCG were cultured in supplemented Middlebrook 7H9 medium (BD Biosciences, Oxford, UK). For infection experiments, mycobacteria were used at midlogarithmic growth at an optical density of 0.60 (Biowave cell density meter; WPA, Cambridge, UK).
Monocyte Purification and Maturation
Monocytes were isolated from single-donor buffy coats (National Blood Transfusion Service, London, UK) by density centrifugation and adhesion purification. Monocytes were matured to monocyte-derived macrophages (macrophages) for 5 d. Medium was then changed to RPMI with 2 mM glutamine and ampicillin (10 μg/ml) and macrophages were infected with MTb or BCG at a multiplicity of infection of 1.
Gene Expression Analysis by Real-Time PolymeraseChain Reaction
Macrophages were lysed with TRI Reagent (Sigma, Poole, UK) and total RNA was extracted. One microgram of RNA was reverse transcribed, using 2 μg of random hexamers (BE Healthcare Bio-Sciences, Little Chalfont, UK) and 200 units of SuperScript II reverse transcriptase (Invitrogen, Paisley, UK), according to the supplier's instructions. Quantitative polymerase chain reactions (PCRs) were done with an ABI PRISM 7700 (Applied Biosystems, Warrington, UK) according to previously described methods (29). The cycle threshold (CT) at which amplification entered the exponential phase was determined and this number was used to indicate the amount of target RNA in each sample.
Casein and Gelatin Zymography
For assessment of MMP-1 and MMP-7 activity, samples were analyzed on 12% casein gels (Invitrogen) and incubated for 40 h in collagenase buffer at 37°C. Caseinolytic activity was revealed by Coomassie blue staining (GE Healthcare, Uppsala, Sweden). All experimental samples were run in parallel with a lane containing 5 ng of recombinant MMP-1 (Calbiochem; Merck Biosciences, Nottingham, UK) to standardize between gels. Densitometric analysis of zymography gels was performed with NIH Image version 1.61 (National Institutes of Health, Bethesda, MD). Gelatin zymography to detect MMP-9 was performed as previously described (30).
Western Blotting for MMP-1 and MMP-7
Western blotting was performed with anti-human MMP-1 antibody (The Binding Site, Birmingham, UK) and MMP-7 Ab-3 (Calbiochem; Merck Biosciences) detected by chemiluminescence.
Immunohistochemistry
Immunohistochemistry for MMP-1 and MMP-7 was performed on paraffin-embedded lung biopsies from six patients with culture-proven MTb infection and six noninfected control subjects. Sections were probed with primary antibodies (MMP-1 Ab-1 [Calbiochem; Merck Biosciences] and MMP-7 Ab-3 [Calbiochem; Merck Biosciences] and isotype controls) and antibody was detected with a non–biotin-based kit (Menarini, Florence, Italy) according to manufacturer's instructions. Consent was obtained from the Hammersmith Hospitals Research Ethics Committee for the use of archived lung biopsies.
Prostaglandin E2 and TIMP-1 ELISAs
Prostaglandin E2 levels in cell culture medium were determined by competitive binding immunoassay (R&D Systems, Abingdon, UK) according to the manufacturer's instructions. The lower level of sensitivity was less than 15.9 pg/ml. TIMP-1 levels in cell culture medium were measured by ELISA (R&D Systems) according to the manufacturer's instructions. The lower level of sensitivity was less than 30 pg/ml.
Statistical Analysis
To analyze the effect of multiple treatments (LPS and MTb) at various times, two-way analysis of variance was performed followed by Tukey's multiple comparison. To analyze the MMP-1 and MMP-7 activity of MTb-infected macrophages compared with control the Student t test was used. A p value of less than 0.05 was taken as statistically significant.
RESULTS
MMP, ADAM, and TIMP Gene Expression in MTb-infected Human Monocyte–derived Macrophages
Gene expression of all 23 human MMPs, nine ADAMs, and four TIMPs was analyzed by reverse transcription (RT)–PCR in unstimulated macrophages and in LPS- and MTb-stimulated macrophages at 6 and 24 h (Figure 1). MTb increased MMP-1 gene expression at 24 h to levels 220-fold greater than control, MMP-3 expression 547-fold greater than control, MMP-7 expression 10-fold greater than control, and MMP-10 expression 114-fold greater than control (all p < 0.05; summarized in Table 1). No significant change in MMP-2, MMP-8, MMP-9, MMP-11, MMP-14, MMP-15, or MMP-17 expression was detected. LPS caused a similar eightfold increase in MMP-7 expression compared with MTb, but increased MMP-1 expression to only 21% of the level stimulated by MTb. LPS also increased MMP-12 expression 15-fold (p < 0.05). MMP-19 to MMP-28 expression was not significantly changed by either stimulus (see Figure E1 in the online supplement). MMP-13, MMP-16, MMP-20, and MMP-26 mRNAs were undetected.
In contrast, MTb did not significantly increase TIMP expression. MTb stimulated a nonsignificant 2.8-fold increase in TIMP-1 expression, whereas TIMP-2, TIMP-3, and TIMP-4 gene expression was suppressed by 2-, 20-, and 2-fold, respectively (Figure 1). Gene expression analysis of the nine ADAMs with a putative protease domain and of the cell-bound MMP inhibitor reversion-inducing cysteine-rich protein with Kazal motifs did not demonstrate any significant changes after MTb or LPS stimulation (see Figures E1 and E2). ADAM-33 was not detected.
The data in Figure 1 present relative mRNA levels normalized to 18S rRNA, indicating the proportional, not absolute, MMP expression level. The CT at which PCR amplification enters the logarithmic phase indicates levels of gene expression; a low threshold reflects high gene expression. Therefore, the CT was used to assess the expression level of each MMP (Figure 2A). Of the MMPs upregulated by MTb, MMP-1 and MMP-7 were the most highly expressed, with mean thresholds in MTb-infected cells of 24.1 and 18.0, respectively, whereas MMP-3 and MMP-10 mRNA levels reached only moderate levels with mean thresholds greater than 25 (Figure 2A). Taken together, the data in Figures 1 and 2A demonstrate that the principal MMPs upregulated by MTb are MMP-1 and MMP-7.
MTb Infection Drives Macrophage MMP-1 andMMP-7 Secretion
We next analyzed MMP secretion to investigate whether increased gene expression results in greater enzyme activity. Casein and gelatin zymography demonstrate that MMP-1, MMP-7, and MMP-9 secretion closely paralleled levels of gene expression (Figure 2B). Specifically, MTb-infected but not LPS-stimulated macrophages secreted detectable levels of MMP-1, whereas both LPS and MTb increased MMP-7 secretion (Figure 2B). High basal MMP-9 secretion was not increased by either stimulus (Figure 2B). TIMP-1 secretion by macrophages measured by ELISA was similarly consistent with gene expression data (data not shown). In kinetic studies, MMP-1 secretion by MTb- infected macrophages increased between 48 and 72 h (Figure 2C). MMP-1 secretion at 72 h was 34.7 ± 7.0 units of caseinolytic activity in MTb-infected macrophages compared with 3.1 ± 0.8 units in control macrophages (Figure 2D; p < 0.05). Macrophages constitutively secreted MMP-7 over 72 h, and this was increased by MTb infection (Figure 2C). There was 3.5-fold greater MMP-7 secretion by infected macrophages compared with control at 48 h and fourfold at 72 h (Figure 2E; p < 0.05). MMP-1 and MMP-7 caseinolytic activity was inhibited completely by 10 μM ethylenediaminetetraacetic acid (data not shown) and Western blotting further confirmed that the zymographic bands were MMP-1 and MMP-7 (Figures 2F and 2G).
Dexamethasone Inhibits MMP Upregulation by MTb
Because dexamethasone is sometimes used clinically to limit tissue damage in severe pulmonary Tb (31) and suppresses MMP-1 upregulation by LPS (32), we investigated the effect of steroid pretreatment on macrophage MMP gene expression and secretion after MTb infection. MMP-1 expression in MTb- infected macrophages was essentially abolished by dexamethasone (Figure 3A) whereas MMP-7 expression was not completely suppressed, being decreased from 3.18 ± 0.57-fold to 1.33 ± 0.35-fold upregulation above control levels (Figure 3B). Similarly, MMP-1 secretion from MTb-infected macrophages was completely inhibited by dexamethasone, whereas MMP-7 secretion was suppressed but remained above basal levels (Figures 3C and 3D).
MMP-1 and MMP-7 Are Expressed in Human Tb Granulomas
To determine the clinicopathologic significance of the cellular findings, lung biopsies from six patients with active culture-proven MTb infection and no evidence of being immunocompromised were stained for MMP-1 and MMP-7 and compared with biopsies from six control uninfected patients (see the online supplement for patient data). Control patients had surgery for malignancy and lung tissue from unaffected areas at the excision margin was examined. In normal lung tissue, MMP-1 and MMP-7 were demonstrated only in alveolar macrophages (Figures 4A and 4B). Macrophages and Langhans' giant cells surrounding the caseous center of the granuloma were strongly immunoreactive for MMP-1 and MMP-7 (Figures 4C and 4D; Figures 4E and 4F, higher magnification). MMP-1 and MMP-7 staining was also demonstrated in more peripheral cells in the granuloma, although this was more evident for MMP-7 (Figure 4D). Specificity was confirmed by the absence of immunoreactivity with the secondary antibody alone or with isotype control primary antibodies (Figures 4G and 4H). Double immunohistochemistry with anti-MMP-1 and anti-CD68 antibodies confirmed that the majority of granuloma cells were immunoreactive for both CD68 and MMP-1, indicating that they were of macrophage lineage (Figure 5).
MMP-1 but Not MMP-7 Gene Expression and Secretion Are MTb Specific and Prostaglandin Dependent
We next investigated potential drivers of MMP-1 and MMP-7 upregulation in MTb infection. Macrophages were infected with MTb or stimulated with IFN- (50 ng/ml), tumor necrosis factor (TNF)- (20 and 50 ng/ml), or interleukin (IL)-1 (50 ng/ml), concentrations previously shown to stimulate MMP-1 secretion in monocytes (33, 34). TNF-, IL-1, and IFN- are pivotal in immune responses to MTb (25, 35–37), but no individual cytokine stimulated significant MMP-1 gene expression or secretion (Figures 6A and 6B). In contrast, MMP-7 gene expression and secretion were increased by TNF- alone (Figures 6A and 6B). IL-1 caused a low-level increase in MMP-7 secretion (Figure 6B). This differential response between MMP-1 and MMP-7 suggested that the upstream regulatory pathways controlling their expression are distinct.
To dissect this divergence, we investigated the prostaglandin pathway, which is key in the control of MMP-1 secretion (22, 38, 39) and has been implicated in differential regulation of MMP-1 and MMP-9 in LPS-stimulated human monocytes (21). MMP-1 secretion in MTb-infected macrophages was inhibited in a dose-dependent manner by preincubation with the cyclooxygenase inhibitor indomethacin. MMP-1 concentrations decreased to control levels after pretreatment with 10 μM indomethacin (Figures 6C and 6D). MMP-7 secretion was not inhibited by 0.1 and 1 μM indomethacin, and was only marginally decreased by 10 μM indomethacin (Figures 6C and 6D).
Virulent MTb Upregulates MMP-1 More Potently Than Vaccine BCG
Because MMP-1 secretion was specifically driven by MTb and not by proinflammatory cytokines, we hypothesized that MMP-1 induction by MTb would be distinct from BCG, the attenuated strain of M. bovis used for vaccination. BCG at a multiplicity of infection of 1 did not cause detectable MMP-1 secretion by macrophages despite prolonged (64 h) casein gel incubation, in contrast to MTb at a multiplicity of infection of 1 (Figure 7A). MMP-1 secretion by BCG-infected cells was also undetectable by Western blotting (data not shown). BCG-stimulated MMP-1 gene expression was at 40% compared with the level found in MTb-infected cells (Figure 7B). In contrast, MMP-7 secretion and gene expression were similar after macrophage infection by either mycobacterium (Figures 7C and 7D).
We investigated the prostaglandin pathway to dissect the mechanism of the pathogen/vaccine divergence further. Prostaglandin E2 accumulation at 24 h in cell culture medium of MTb-infected macrophages was threefold greater than that after BCG infection (Figure 7E). To determine whether prostaglandin pathway supplementation could drive MMP-1 secretion in BCG-infected macrophages, the downstream prostaglandin E2 pathway was augmented with dibutyryl cAMP (B2cAMP), a synthetic analog of cAMP. Stimulation of BCG-infected macrophages with B2cAMP increased MMP-1 secretion (Figure 7F) in a bell-shaped response. B2cAMP alone did not alter MMP-1 secretion (data not shown). Thus, divergent MMP-1 secretion by macrophages in response to MTb and BCG stimulation is in part due to differential prostaglandin E2 synthesis affecting MMP-1 gene expression.
DISCUSSION
Pulmonary cavitation is fundamental to the global success of MTb, as it facilitates mycobacterial transmission to new hosts. The biochemistry of the lung matrix predicts the involvement of collagenases and elastases in tissue destruction. In a comprehensive analysis of MMP, ADAM, and TIMP gene expression in human macrophages, we demonstrate that MTb infection potently upregulates MMP-1 and MMP-7 expression. No concurrent significant increase in TIMP-1 to TIMP-4 expression was found. Increased gene expression drives MMP-1 and MMP-7 secretion. The upregulation of these specific MMPs by MTb may be critical to pathology, as they degrade key components of the lung matrix. MMP-1 activity is rate limiting in the degradation of type I collagen (40), the primary architectural collagen of the lung (41). Expression levels of other collagen-degrading enzymes, specifically MMP-2, MMP-8, MMP-9, MMP-13, and MMP-14, were not elevated after MTb infection, implicating MMP-1 as the principal collagenase in this pathology. The second enzyme upregulated by MTb, MMP-7, efficiently cleaves elastin (16), which is usually highly metabolically stable and lasts life-long (42). MMP-3 and MMP-10 are also induced by MTb, although to lower levels of expression as determined by the PCR cycle threshold. They may cleave cross-linking fibrils, thereby unmasking primary fibers and permitting their degradation (43). Thus MTb promotes the breakdown of collagen and elastin, an essential step in pulmonary cavity formation.
Excess MMP activity is implicated in other destructive pulmonary pathologies in both human and animal studies (10–13, 44–47). Increased MMP-1 expression is found in the macrophages and lungs of patients with emphysema and also in animal models (44–47). Transgenic mice overexpressing MMP-1 spontaneously develop emphysema (10). Interestingly, the proposed mouse ortholog of MMP-1, Mcol-A, has greatly reduced activity against fibrillar type I collagen compared with human MMP-1 (48). Thus different collagenolytic activity may in part explain the divergent pathologies of human and mouse Tb (5, 6).
MMP induction by mycobacteria has been described but until now no global analysis of MMP gene expression after MTb infection has been performed. The key role of MMP-1 is consistent with the observation that the water-soluble fraction of Mycobacterium smegmatis increases collagenase synthesis by guinea pig peritoneal macrophages (49), and human THP-1 cells stimulated by mycobacterial lipoarabinomannan upregulate MMP-1 expression (50). No previous studies have investigated MMP-7 in MTb infection. MTb-dependent expression of MMP-9 is described in murine macrophages, human THP-1 cells, and primary monocytes (50–53) and we described relatively unopposed MMP-9 expression in tuberculous lymph nodes (54). However, in macrocyte colony-stimulating factor–matured macrophages we observed high constitutive MMP-9 expression that did not increase with MTb infection, illustrating the critical role of cellular differentiation in MMP regulation (19, 20, 55). We demonstrated a tendency to decreased MMP-9 expression in LPS-stimulated macrophages, which is consistent with a report of LPS suppressing MMP-9 secretion by stimulated monocytes (20). However, our data diverge from other experiments in which LPS upregulates MMP-9 secretion (21). Such contrasting findings may result from different monocyte maturation and the presence of LPS-binding protein in cell culture medium.
Our cellular model predicted high-level MMP-1 and MMP-7 expression at the site of tissue destruction and this was observed in clinical biopsy specimens from patients undergoing disease reactivation, where MTb will be actively replicating. Macrophage markers and MMP-1 colocalized and macrophages may be the critical mediators of tissue destruction in Tb (26). These data suggest that macrophages, as the effector cells of the immune response, may drive pulmonary cavitation, which was thought to require an acquired immune response (56). However, in guinea pigs caseating necrosis and matrix destruction develop before the acquired immune response, suggesting that innate mechanisms cause immunopathology (57).
Clinically, dexamethasone is used to limit immunopathology in Tb (58) and, although never tested by a randomized clinical trial, is sometimes used as adjunctive therapy in severe pulmonary Tb (31). Therefore, its effect on MMP-1 and MMP-7 expression in MTb-infected macrophages is of interest. Dexamethasone completely suppresses MMP-1 and reduces MMP-7 secretion in infected macrophages, consistent with a report of dexamethasone suppressing alveolar macrophage MMP-1 expression after LPS stimulation (32). Dexamethasone suppresses MMP expression at the level of transcription (59), and this is a potential mechanism whereby steroids limit tissue damage in Tb. The MMP-1 effect may be particularly important because our data strongly suggest that MTb is a specific stimulus to MMP-1 secretion.
MTb, but not important proinflammatory cytokines involved in orchestrating the host immune response, increased MMP-1 secretion from macrophages whereas MMP-7 expression and secretion could be stimulated by TNF- alone. In vivo, macrophage MMP expression will be modulated by a complex interaction between MTb infection and host cytokines, which may further increase or suppress expression. Increased MMP-1 expression has been shown to require multiple stimuli in both monocytes and macrophages, which have divergent MMP expression (33, 34, 60), but our data differ from a report of TNF- not upregulating MMP-7 secretion (23). This may result from the specific macrophage maturation protocols used or the greater sensitivity of RT-PCR over Western analysis in detecting MMP-7 upregulation.
The divergent MMP-1 and MMP-7 response to TNF- and steroid treatment suggested that the upstream regulation of these MMPs differs. MTb-induced MMP-1 secretion in macrophages was regulated by a prostaglandin-dependent pathway, but MMP-7 secretion was prostaglandin independent. Collagenase secretion by guinea pig macrophages stimulated with M. smegmatis extract was also prostaglandin dependent (49). MMP-7 regulation in MTb appears different from a report of prostaglandin-dependent LPS-stimulated MMP-7 expression in macrophages (23), indicating stimulus-specific MMP regulation.
BCG, the Tb vaccine, does not cause pulmonary cavitation in the immunocompetent host and did not upregulate MMP-1 gene expression and secretion as potently as MTb, whereas MMP-7 gene expression and secretion subsequent to both stimuli are similar. Although BCG induced MMP-1 gene expression to 40% compared with the level in MTb-infected monocyte-derived macrophages, MMP-1 secretion was undetectable despite prolonged gel incubation to increase sensitivity. This suggests that further factors in addition to reduced mRNA expression contribute to decreased secretion. The genome analysis of BCG demonstrates that an entire DNA region (RD1) is deleted (61), encoding key antigens such as ESAT-6, which may be key to MMP upregulation. The difference between MTb and BCG is in part explained by differential prostaglandin E2 secretion by infected macrophages. Supplementation with B2cAMP augments MMP-1 secretion in BCG-infected macrophages but does not increase secretion as a single stimulus, indicating that the prostaglandin pathway is necessary but not sufficient for MMP-1 secretion. Others have also found that augmenting the prostaglandin pathway alone does not drive MMP-1 secretion (39). In murine macrophages infected with Mycobacterium avium intracellulare, prostaglandin E2 production correlates with virulence (62). Excess prostaglandin activity is implicated in other tissue-destructive pathologies, such as periodontal disease, in which pharmacologic suppression of the prostaglandin pathway can limit pathology (63). Our data implicate prostaglandin E2–dependent MMP-1 secretion as an important step in the pathogenesis of Tb.
In summary, we demonstrate that MTb potently upregulates MMP-1 and MMP-7 expression in human macrophages. In the lungs of patients with Tb, macrophages express MMP-1 and MMP-7 at the site of tissue destruction. MMP-1 upregulation is MTb specific and regulated by prostaglandin E2. The vaccine BCG, which does not cause significant tissue damage, fails to upregulate MMP-1 equivalently to MTb, although both cause equal increases in MMP-7 gene expression and secretion. This pathogen–vaccine divergence is regulated by prostaglandin E2 production. Together with evidence linking MMP-1 with pulmonary destruction in other diseases (10–13, 44–47), our data suggest that increased MMP-1 expression in MTb-infected macrophages is a key step in cavity formation. Modulating excessive MMP-1 activity is a potential therapeutic target to reduce immunopathology in Tb.
Acknowledgments
The authors thank Aileen Hogan for designing the ADAM primer and probes.
FOOTNOTES
These authors contributed equally to this work.
Supported by a Wellcome Trust Clinical Research Training Fellowship (P.E.), the Hammersmith Hospitals Trustees' Research Committee, and by a grant from the European Union Framework Program 6 (LSHC-CT-2003-503297).
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1164/rccm.200505-753OC on September 1, 2005
Conflict of Interest Statement: None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
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School of Biological Sciences, University of East Anglia, Norwich, United Kingdom
ABSTRACT
Rationale: Pulmonary cavitation is fundamental to the global success of Mycobacterium tuberculosis. However, the mechanisms of this lung destruction are poorly understood. The biochemistry of lung matrix predicts matrix metalloproteinase (MMP) involvement in immunopathology.
Methods: We investigated gene expression of all MMPs, proteins with a disintegrin and metalloproteinase domain, and tissue inhibitors of metalloproteinases in M. tuberculosis–infected human macrophages by real-time polymerase chain reaction. MMP secretion was measured by zymography and Western analysis, and expression in patients with pulmonary tuberculosis was localized by immunohistochemistry.
Results: MMP-1 and MMP-7 gene expression and secretion are potently upregulated by M. tuberculosis, and no increase in tissue inhibitor of metalloproteinase expression occurs to oppose their activity. Dexamethasone completely suppresses MMP-1 but not MMP-7 gene expression and secretion. In patients with active tuberculosis, macrophages express MMP-1 and MMP-7 adjacent to areas of tissue destruction. MMP-1 but not MMP-7 expression and secretion are relatively M. tuberculosis specific, are not upregulated by tuberculosis- associated cytokines, and are prostaglandin dependent. In contrast, the vaccine M. bovis bacillus Calmette-Guerin (BCG) does not stimulate MMP-1 secretion from human macrophages, although M. tuberculosis and BCG do upregulate MMP-7 equally. BCG-infected macrophages secrete reduced prostaglandin E2 concentrations compared with M. tuberculosis–infected macrophages, and prostaglandin pathway supplementation augments MMP-1 secretion from BCG-infected cells.
Conclusions: M. tuberculosis specifically upregulates MMP-1 in a cellular model of human infection and in patients with tuberculosis. In contrast, vaccine BCG, which does not cause lung cavitation, does not upregulate prostaglandin E2–dependent MMP-1 secretion.
Key Words: macrophage matrix metalloproteinases Mycobacterium tuberculosis pathology prostaglandin E
Mycobacterium tuberculosis (MTb) infects approximately one-third of the world's population and kills 2 to 3 million people each year (1, 2). Pulmonary cavitation is vital to the transmission of MTb and favors bacterial persistence (3). The cavity is an immunoprivileged site, with MTb replicating freely in the cavity wall (3). After treatment of tuberculosis (Tb), opportunistic pathogens may colonize the residual cavity (4). The ability to cause pulmonary cavitation in the immunocompetent host differentiates MTb from the vaccine strain Mycobacterium bovis bacillus Calmette-Guerin (BCG). The standard animal model of Tb is the mouse, which is valuable in studying immunity to MTb (5), but the pathology of murine Tb is divergent from that of human Tb, being purely fibrotic without cavitation (5, 6). Guinea pigs develop granulomas similar to those in humans but rarely cavitate, whereas rabbits develop cavitation with BCG, which is not a pulmonary pathogen in humans (5). Therefore, mechanisms by which MTb causes tissue destruction are best investigated in primary human cells and by clinical studies.
Proteases from inflammatory cells have been hypothesized to cause tissue destruction in Tb since the era of Koch and Virchow (7). For cavitation to occur, the extracellular matrix of the lung must be degraded. Fibrillar type I collagen provides the tensile strength of the matrix and elastin allows distensibility (8). Type I collagen is highly resistant to enzymatic degradation and only matrix metalloproteinases (MMPs) are able to cleave it at neutral pH (9). Excess MMP activity in the lung causes tissue destruction. For example, mice constitutively overexpressing MMP-1 (interstitial collagenase) develop emphysema (10) and MMPs are implicated in other animal models of emphysema (11–13).
MMPs are a family of zinc-binding proteases that collectively degrade all components of the extracellular matrix (14) and are from the metalloprotease class that includes the family of proteins with a disintegrin and metalloproteinase domain (ADAMs). MMP-1, MMP-8, and MMP-13 are the primary enzymes responsible for degradation of type I collagen (15). Multiple MMPs can degrade elastin, including MMP-2, MMP-7, MMP-9, and MMP-12 (15). MMP-7 (matrilysin) is the most potent elastase produced by macrophages (16). In addition to matrix remodeling, MMPs have important roles in immunity, such as the proteolytic processing of chemokines and cytokines (17, 18). MMP activity is regulated by multiple mechanisms, including transcriptional control, secretion as proenzymes that require proteolytic activation, compartmentalization, and secretion of specific inhibitors (tissue inhibitors of metalloproteinases [TIMPs]) (17).
MMP expression changes with cellular differentiation: macrophages secrete a broader spectrum and greater quantities of these enzymes than do monocytes (19, 20). Macrophage MMP production is regulated primarily through a pathway dependent on prostaglandin E2 and cAMP (21–23). Macrophages are a principal source of MMPs at inflammatory loci (24). In Tb, macrophages form the central core of the granuloma and are the key effector of the host immune response to MTb (25). In pulmonary MTb infection, excessive proteolytic activity causes matrix breakdown, caseation of the granuloma, liquefaction, and finally cavitation (26). We hypothesize that MMPs are critical to this pulmonary destruction in Tb.
This work has been previously presented in part in the form of abstracts (27, 28).
METHODS
See the online supplement for additional details of the following methods.
MTb and BCG Culture
M. tuberculosis H37Rv and M. bovis BCG were cultured in supplemented Middlebrook 7H9 medium (BD Biosciences, Oxford, UK). For infection experiments, mycobacteria were used at midlogarithmic growth at an optical density of 0.60 (Biowave cell density meter; WPA, Cambridge, UK).
Monocyte Purification and Maturation
Monocytes were isolated from single-donor buffy coats (National Blood Transfusion Service, London, UK) by density centrifugation and adhesion purification. Monocytes were matured to monocyte-derived macrophages (macrophages) for 5 d. Medium was then changed to RPMI with 2 mM glutamine and ampicillin (10 μg/ml) and macrophages were infected with MTb or BCG at a multiplicity of infection of 1.
Gene Expression Analysis by Real-Time PolymeraseChain Reaction
Macrophages were lysed with TRI Reagent (Sigma, Poole, UK) and total RNA was extracted. One microgram of RNA was reverse transcribed, using 2 μg of random hexamers (BE Healthcare Bio-Sciences, Little Chalfont, UK) and 200 units of SuperScript II reverse transcriptase (Invitrogen, Paisley, UK), according to the supplier's instructions. Quantitative polymerase chain reactions (PCRs) were done with an ABI PRISM 7700 (Applied Biosystems, Warrington, UK) according to previously described methods (29). The cycle threshold (CT) at which amplification entered the exponential phase was determined and this number was used to indicate the amount of target RNA in each sample.
Casein and Gelatin Zymography
For assessment of MMP-1 and MMP-7 activity, samples were analyzed on 12% casein gels (Invitrogen) and incubated for 40 h in collagenase buffer at 37°C. Caseinolytic activity was revealed by Coomassie blue staining (GE Healthcare, Uppsala, Sweden). All experimental samples were run in parallel with a lane containing 5 ng of recombinant MMP-1 (Calbiochem; Merck Biosciences, Nottingham, UK) to standardize between gels. Densitometric analysis of zymography gels was performed with NIH Image version 1.61 (National Institutes of Health, Bethesda, MD). Gelatin zymography to detect MMP-9 was performed as previously described (30).
Western Blotting for MMP-1 and MMP-7
Western blotting was performed with anti-human MMP-1 antibody (The Binding Site, Birmingham, UK) and MMP-7 Ab-3 (Calbiochem; Merck Biosciences) detected by chemiluminescence.
Immunohistochemistry
Immunohistochemistry for MMP-1 and MMP-7 was performed on paraffin-embedded lung biopsies from six patients with culture-proven MTb infection and six noninfected control subjects. Sections were probed with primary antibodies (MMP-1 Ab-1 [Calbiochem; Merck Biosciences] and MMP-7 Ab-3 [Calbiochem; Merck Biosciences] and isotype controls) and antibody was detected with a non–biotin-based kit (Menarini, Florence, Italy) according to manufacturer's instructions. Consent was obtained from the Hammersmith Hospitals Research Ethics Committee for the use of archived lung biopsies.
Prostaglandin E2 and TIMP-1 ELISAs
Prostaglandin E2 levels in cell culture medium were determined by competitive binding immunoassay (R&D Systems, Abingdon, UK) according to the manufacturer's instructions. The lower level of sensitivity was less than 15.9 pg/ml. TIMP-1 levels in cell culture medium were measured by ELISA (R&D Systems) according to the manufacturer's instructions. The lower level of sensitivity was less than 30 pg/ml.
Statistical Analysis
To analyze the effect of multiple treatments (LPS and MTb) at various times, two-way analysis of variance was performed followed by Tukey's multiple comparison. To analyze the MMP-1 and MMP-7 activity of MTb-infected macrophages compared with control the Student t test was used. A p value of less than 0.05 was taken as statistically significant.
RESULTS
MMP, ADAM, and TIMP Gene Expression in MTb-infected Human Monocyte–derived Macrophages
Gene expression of all 23 human MMPs, nine ADAMs, and four TIMPs was analyzed by reverse transcription (RT)–PCR in unstimulated macrophages and in LPS- and MTb-stimulated macrophages at 6 and 24 h (Figure 1). MTb increased MMP-1 gene expression at 24 h to levels 220-fold greater than control, MMP-3 expression 547-fold greater than control, MMP-7 expression 10-fold greater than control, and MMP-10 expression 114-fold greater than control (all p < 0.05; summarized in Table 1). No significant change in MMP-2, MMP-8, MMP-9, MMP-11, MMP-14, MMP-15, or MMP-17 expression was detected. LPS caused a similar eightfold increase in MMP-7 expression compared with MTb, but increased MMP-1 expression to only 21% of the level stimulated by MTb. LPS also increased MMP-12 expression 15-fold (p < 0.05). MMP-19 to MMP-28 expression was not significantly changed by either stimulus (see Figure E1 in the online supplement). MMP-13, MMP-16, MMP-20, and MMP-26 mRNAs were undetected.
In contrast, MTb did not significantly increase TIMP expression. MTb stimulated a nonsignificant 2.8-fold increase in TIMP-1 expression, whereas TIMP-2, TIMP-3, and TIMP-4 gene expression was suppressed by 2-, 20-, and 2-fold, respectively (Figure 1). Gene expression analysis of the nine ADAMs with a putative protease domain and of the cell-bound MMP inhibitor reversion-inducing cysteine-rich protein with Kazal motifs did not demonstrate any significant changes after MTb or LPS stimulation (see Figures E1 and E2). ADAM-33 was not detected.
The data in Figure 1 present relative mRNA levels normalized to 18S rRNA, indicating the proportional, not absolute, MMP expression level. The CT at which PCR amplification enters the logarithmic phase indicates levels of gene expression; a low threshold reflects high gene expression. Therefore, the CT was used to assess the expression level of each MMP (Figure 2A). Of the MMPs upregulated by MTb, MMP-1 and MMP-7 were the most highly expressed, with mean thresholds in MTb-infected cells of 24.1 and 18.0, respectively, whereas MMP-3 and MMP-10 mRNA levels reached only moderate levels with mean thresholds greater than 25 (Figure 2A). Taken together, the data in Figures 1 and 2A demonstrate that the principal MMPs upregulated by MTb are MMP-1 and MMP-7.
MTb Infection Drives Macrophage MMP-1 andMMP-7 Secretion
We next analyzed MMP secretion to investigate whether increased gene expression results in greater enzyme activity. Casein and gelatin zymography demonstrate that MMP-1, MMP-7, and MMP-9 secretion closely paralleled levels of gene expression (Figure 2B). Specifically, MTb-infected but not LPS-stimulated macrophages secreted detectable levels of MMP-1, whereas both LPS and MTb increased MMP-7 secretion (Figure 2B). High basal MMP-9 secretion was not increased by either stimulus (Figure 2B). TIMP-1 secretion by macrophages measured by ELISA was similarly consistent with gene expression data (data not shown). In kinetic studies, MMP-1 secretion by MTb- infected macrophages increased between 48 and 72 h (Figure 2C). MMP-1 secretion at 72 h was 34.7 ± 7.0 units of caseinolytic activity in MTb-infected macrophages compared with 3.1 ± 0.8 units in control macrophages (Figure 2D; p < 0.05). Macrophages constitutively secreted MMP-7 over 72 h, and this was increased by MTb infection (Figure 2C). There was 3.5-fold greater MMP-7 secretion by infected macrophages compared with control at 48 h and fourfold at 72 h (Figure 2E; p < 0.05). MMP-1 and MMP-7 caseinolytic activity was inhibited completely by 10 μM ethylenediaminetetraacetic acid (data not shown) and Western blotting further confirmed that the zymographic bands were MMP-1 and MMP-7 (Figures 2F and 2G).
Dexamethasone Inhibits MMP Upregulation by MTb
Because dexamethasone is sometimes used clinically to limit tissue damage in severe pulmonary Tb (31) and suppresses MMP-1 upregulation by LPS (32), we investigated the effect of steroid pretreatment on macrophage MMP gene expression and secretion after MTb infection. MMP-1 expression in MTb- infected macrophages was essentially abolished by dexamethasone (Figure 3A) whereas MMP-7 expression was not completely suppressed, being decreased from 3.18 ± 0.57-fold to 1.33 ± 0.35-fold upregulation above control levels (Figure 3B). Similarly, MMP-1 secretion from MTb-infected macrophages was completely inhibited by dexamethasone, whereas MMP-7 secretion was suppressed but remained above basal levels (Figures 3C and 3D).
MMP-1 and MMP-7 Are Expressed in Human Tb Granulomas
To determine the clinicopathologic significance of the cellular findings, lung biopsies from six patients with active culture-proven MTb infection and no evidence of being immunocompromised were stained for MMP-1 and MMP-7 and compared with biopsies from six control uninfected patients (see the online supplement for patient data). Control patients had surgery for malignancy and lung tissue from unaffected areas at the excision margin was examined. In normal lung tissue, MMP-1 and MMP-7 were demonstrated only in alveolar macrophages (Figures 4A and 4B). Macrophages and Langhans' giant cells surrounding the caseous center of the granuloma were strongly immunoreactive for MMP-1 and MMP-7 (Figures 4C and 4D; Figures 4E and 4F, higher magnification). MMP-1 and MMP-7 staining was also demonstrated in more peripheral cells in the granuloma, although this was more evident for MMP-7 (Figure 4D). Specificity was confirmed by the absence of immunoreactivity with the secondary antibody alone or with isotype control primary antibodies (Figures 4G and 4H). Double immunohistochemistry with anti-MMP-1 and anti-CD68 antibodies confirmed that the majority of granuloma cells were immunoreactive for both CD68 and MMP-1, indicating that they were of macrophage lineage (Figure 5).
MMP-1 but Not MMP-7 Gene Expression and Secretion Are MTb Specific and Prostaglandin Dependent
We next investigated potential drivers of MMP-1 and MMP-7 upregulation in MTb infection. Macrophages were infected with MTb or stimulated with IFN- (50 ng/ml), tumor necrosis factor (TNF)- (20 and 50 ng/ml), or interleukin (IL)-1 (50 ng/ml), concentrations previously shown to stimulate MMP-1 secretion in monocytes (33, 34). TNF-, IL-1, and IFN- are pivotal in immune responses to MTb (25, 35–37), but no individual cytokine stimulated significant MMP-1 gene expression or secretion (Figures 6A and 6B). In contrast, MMP-7 gene expression and secretion were increased by TNF- alone (Figures 6A and 6B). IL-1 caused a low-level increase in MMP-7 secretion (Figure 6B). This differential response between MMP-1 and MMP-7 suggested that the upstream regulatory pathways controlling their expression are distinct.
To dissect this divergence, we investigated the prostaglandin pathway, which is key in the control of MMP-1 secretion (22, 38, 39) and has been implicated in differential regulation of MMP-1 and MMP-9 in LPS-stimulated human monocytes (21). MMP-1 secretion in MTb-infected macrophages was inhibited in a dose-dependent manner by preincubation with the cyclooxygenase inhibitor indomethacin. MMP-1 concentrations decreased to control levels after pretreatment with 10 μM indomethacin (Figures 6C and 6D). MMP-7 secretion was not inhibited by 0.1 and 1 μM indomethacin, and was only marginally decreased by 10 μM indomethacin (Figures 6C and 6D).
Virulent MTb Upregulates MMP-1 More Potently Than Vaccine BCG
Because MMP-1 secretion was specifically driven by MTb and not by proinflammatory cytokines, we hypothesized that MMP-1 induction by MTb would be distinct from BCG, the attenuated strain of M. bovis used for vaccination. BCG at a multiplicity of infection of 1 did not cause detectable MMP-1 secretion by macrophages despite prolonged (64 h) casein gel incubation, in contrast to MTb at a multiplicity of infection of 1 (Figure 7A). MMP-1 secretion by BCG-infected cells was also undetectable by Western blotting (data not shown). BCG-stimulated MMP-1 gene expression was at 40% compared with the level found in MTb-infected cells (Figure 7B). In contrast, MMP-7 secretion and gene expression were similar after macrophage infection by either mycobacterium (Figures 7C and 7D).
We investigated the prostaglandin pathway to dissect the mechanism of the pathogen/vaccine divergence further. Prostaglandin E2 accumulation at 24 h in cell culture medium of MTb-infected macrophages was threefold greater than that after BCG infection (Figure 7E). To determine whether prostaglandin pathway supplementation could drive MMP-1 secretion in BCG-infected macrophages, the downstream prostaglandin E2 pathway was augmented with dibutyryl cAMP (B2cAMP), a synthetic analog of cAMP. Stimulation of BCG-infected macrophages with B2cAMP increased MMP-1 secretion (Figure 7F) in a bell-shaped response. B2cAMP alone did not alter MMP-1 secretion (data not shown). Thus, divergent MMP-1 secretion by macrophages in response to MTb and BCG stimulation is in part due to differential prostaglandin E2 synthesis affecting MMP-1 gene expression.
DISCUSSION
Pulmonary cavitation is fundamental to the global success of MTb, as it facilitates mycobacterial transmission to new hosts. The biochemistry of the lung matrix predicts the involvement of collagenases and elastases in tissue destruction. In a comprehensive analysis of MMP, ADAM, and TIMP gene expression in human macrophages, we demonstrate that MTb infection potently upregulates MMP-1 and MMP-7 expression. No concurrent significant increase in TIMP-1 to TIMP-4 expression was found. Increased gene expression drives MMP-1 and MMP-7 secretion. The upregulation of these specific MMPs by MTb may be critical to pathology, as they degrade key components of the lung matrix. MMP-1 activity is rate limiting in the degradation of type I collagen (40), the primary architectural collagen of the lung (41). Expression levels of other collagen-degrading enzymes, specifically MMP-2, MMP-8, MMP-9, MMP-13, and MMP-14, were not elevated after MTb infection, implicating MMP-1 as the principal collagenase in this pathology. The second enzyme upregulated by MTb, MMP-7, efficiently cleaves elastin (16), which is usually highly metabolically stable and lasts life-long (42). MMP-3 and MMP-10 are also induced by MTb, although to lower levels of expression as determined by the PCR cycle threshold. They may cleave cross-linking fibrils, thereby unmasking primary fibers and permitting their degradation (43). Thus MTb promotes the breakdown of collagen and elastin, an essential step in pulmonary cavity formation.
Excess MMP activity is implicated in other destructive pulmonary pathologies in both human and animal studies (10–13, 44–47). Increased MMP-1 expression is found in the macrophages and lungs of patients with emphysema and also in animal models (44–47). Transgenic mice overexpressing MMP-1 spontaneously develop emphysema (10). Interestingly, the proposed mouse ortholog of MMP-1, Mcol-A, has greatly reduced activity against fibrillar type I collagen compared with human MMP-1 (48). Thus different collagenolytic activity may in part explain the divergent pathologies of human and mouse Tb (5, 6).
MMP induction by mycobacteria has been described but until now no global analysis of MMP gene expression after MTb infection has been performed. The key role of MMP-1 is consistent with the observation that the water-soluble fraction of Mycobacterium smegmatis increases collagenase synthesis by guinea pig peritoneal macrophages (49), and human THP-1 cells stimulated by mycobacterial lipoarabinomannan upregulate MMP-1 expression (50). No previous studies have investigated MMP-7 in MTb infection. MTb-dependent expression of MMP-9 is described in murine macrophages, human THP-1 cells, and primary monocytes (50–53) and we described relatively unopposed MMP-9 expression in tuberculous lymph nodes (54). However, in macrocyte colony-stimulating factor–matured macrophages we observed high constitutive MMP-9 expression that did not increase with MTb infection, illustrating the critical role of cellular differentiation in MMP regulation (19, 20, 55). We demonstrated a tendency to decreased MMP-9 expression in LPS-stimulated macrophages, which is consistent with a report of LPS suppressing MMP-9 secretion by stimulated monocytes (20). However, our data diverge from other experiments in which LPS upregulates MMP-9 secretion (21). Such contrasting findings may result from different monocyte maturation and the presence of LPS-binding protein in cell culture medium.
Our cellular model predicted high-level MMP-1 and MMP-7 expression at the site of tissue destruction and this was observed in clinical biopsy specimens from patients undergoing disease reactivation, where MTb will be actively replicating. Macrophage markers and MMP-1 colocalized and macrophages may be the critical mediators of tissue destruction in Tb (26). These data suggest that macrophages, as the effector cells of the immune response, may drive pulmonary cavitation, which was thought to require an acquired immune response (56). However, in guinea pigs caseating necrosis and matrix destruction develop before the acquired immune response, suggesting that innate mechanisms cause immunopathology (57).
Clinically, dexamethasone is used to limit immunopathology in Tb (58) and, although never tested by a randomized clinical trial, is sometimes used as adjunctive therapy in severe pulmonary Tb (31). Therefore, its effect on MMP-1 and MMP-7 expression in MTb-infected macrophages is of interest. Dexamethasone completely suppresses MMP-1 and reduces MMP-7 secretion in infected macrophages, consistent with a report of dexamethasone suppressing alveolar macrophage MMP-1 expression after LPS stimulation (32). Dexamethasone suppresses MMP expression at the level of transcription (59), and this is a potential mechanism whereby steroids limit tissue damage in Tb. The MMP-1 effect may be particularly important because our data strongly suggest that MTb is a specific stimulus to MMP-1 secretion.
MTb, but not important proinflammatory cytokines involved in orchestrating the host immune response, increased MMP-1 secretion from macrophages whereas MMP-7 expression and secretion could be stimulated by TNF- alone. In vivo, macrophage MMP expression will be modulated by a complex interaction between MTb infection and host cytokines, which may further increase or suppress expression. Increased MMP-1 expression has been shown to require multiple stimuli in both monocytes and macrophages, which have divergent MMP expression (33, 34, 60), but our data differ from a report of TNF- not upregulating MMP-7 secretion (23). This may result from the specific macrophage maturation protocols used or the greater sensitivity of RT-PCR over Western analysis in detecting MMP-7 upregulation.
The divergent MMP-1 and MMP-7 response to TNF- and steroid treatment suggested that the upstream regulation of these MMPs differs. MTb-induced MMP-1 secretion in macrophages was regulated by a prostaglandin-dependent pathway, but MMP-7 secretion was prostaglandin independent. Collagenase secretion by guinea pig macrophages stimulated with M. smegmatis extract was also prostaglandin dependent (49). MMP-7 regulation in MTb appears different from a report of prostaglandin-dependent LPS-stimulated MMP-7 expression in macrophages (23), indicating stimulus-specific MMP regulation.
BCG, the Tb vaccine, does not cause pulmonary cavitation in the immunocompetent host and did not upregulate MMP-1 gene expression and secretion as potently as MTb, whereas MMP-7 gene expression and secretion subsequent to both stimuli are similar. Although BCG induced MMP-1 gene expression to 40% compared with the level in MTb-infected monocyte-derived macrophages, MMP-1 secretion was undetectable despite prolonged gel incubation to increase sensitivity. This suggests that further factors in addition to reduced mRNA expression contribute to decreased secretion. The genome analysis of BCG demonstrates that an entire DNA region (RD1) is deleted (61), encoding key antigens such as ESAT-6, which may be key to MMP upregulation. The difference between MTb and BCG is in part explained by differential prostaglandin E2 secretion by infected macrophages. Supplementation with B2cAMP augments MMP-1 secretion in BCG-infected macrophages but does not increase secretion as a single stimulus, indicating that the prostaglandin pathway is necessary but not sufficient for MMP-1 secretion. Others have also found that augmenting the prostaglandin pathway alone does not drive MMP-1 secretion (39). In murine macrophages infected with Mycobacterium avium intracellulare, prostaglandin E2 production correlates with virulence (62). Excess prostaglandin activity is implicated in other tissue-destructive pathologies, such as periodontal disease, in which pharmacologic suppression of the prostaglandin pathway can limit pathology (63). Our data implicate prostaglandin E2–dependent MMP-1 secretion as an important step in the pathogenesis of Tb.
In summary, we demonstrate that MTb potently upregulates MMP-1 and MMP-7 expression in human macrophages. In the lungs of patients with Tb, macrophages express MMP-1 and MMP-7 at the site of tissue destruction. MMP-1 upregulation is MTb specific and regulated by prostaglandin E2. The vaccine BCG, which does not cause significant tissue damage, fails to upregulate MMP-1 equivalently to MTb, although both cause equal increases in MMP-7 gene expression and secretion. This pathogen–vaccine divergence is regulated by prostaglandin E2 production. Together with evidence linking MMP-1 with pulmonary destruction in other diseases (10–13, 44–47), our data suggest that increased MMP-1 expression in MTb-infected macrophages is a key step in cavity formation. Modulating excessive MMP-1 activity is a potential therapeutic target to reduce immunopathology in Tb.
Acknowledgments
The authors thank Aileen Hogan for designing the ADAM primer and probes.
FOOTNOTES
These authors contributed equally to this work.
Supported by a Wellcome Trust Clinical Research Training Fellowship (P.E.), the Hammersmith Hospitals Trustees' Research Committee, and by a grant from the European Union Framework Program 6 (LSHC-CT-2003-503297).
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1164/rccm.200505-753OC on September 1, 2005
Conflict of Interest Statement: None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
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