help button home button
AJRCCM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on September 1, 2005, doi:10.1164/rccm.200505-753OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Online Supplement
Right arrow All Versions of this Article:
200505-753OCv1
172/12/1596    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Elkington, P. T. G.
Right arrow Articles by Friedland, J. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Elkington, P. T. G.
Right arrow Articles by Friedland, J. S.
American Journal of Respiratory and Critical Care Medicine Vol 172. pp. 1596-1604, (2005)
© 2005 American Thoracic Society
doi: 10.1164/rccm.200505-753OC

Original Article

Mycobacterium tuberculosis, but Not Vaccine BCG, Specifically Upregulates Matrix Metalloproteinase-1

Paul T. G. Elkington*, Robert K. Nuttall*, Joseph J. Boyle, Cecilia M. O'Kane, Donna E. Horncastle, Dylan R. Edwards and Jon S. Friedland

Department of Infectious Diseases and Department of Histopathology, Imperial College, Hammersmith Campus, London; and School of Biological Sciences, University of East Anglia, Norwich, United Kingdom

Correspondence and requests for reprints should be addressed to J. S. Friedland, Ph.D., Department of Infectious Diseases, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK. E-mail: j.friedland{at}imperial.ac.uk


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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-Guérin (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-Guérin (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
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
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
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
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.


View this table:
[in this window]
[in a new window]
  		TABLE 1. EXPRESSION OF GENES ENCODING MATRIX METALLOPROTEINASES, PROTEINS WITH A DISINTEGRIN AND METALLOPROTEINASE DOMAIN, AND TISSUE INHIBITORS OF METALLOPROTEINASES IN CONTROL AND LPS- AND Mycobacterium tuberculosis-STIMULATED HUMAN MACROPHAGES

 
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.



View larger version (30K):
[in this window]
[in a new window]
  		Figure 1. Matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinases (TIMP) gene expression in human macrophages stimulated by LPS and Mycobacterium tuberculosis (MTb). Macrophages were stimulated with LPS (1 µg/ml) or MTb at a multiplicity of infection (MOI) of 1. Total RNA was extracted at 6 and 24 h, and mRNA accumulation was analyzed by reverse transcriptase–polymerase chain reaction (RT-PCR). For each gene, mRNA levels normalized to 18S RNA are expressed as fold change relative to control mRNA levels at 6 h. All 23 human MMPs, nine ADAMs, and four TIMPs were analyzed. Open bars depict control, shaded bars depict LPS-stimulated, and solid bars depict MTb-stimulated macrophage gene expression. MTb stimulated significant increases in MMP-1, MMP-3, MMP-7, and MMP-10 gene expression. LPS stimulated significant increases in MMP-7 and MMP-12 expression. MMP-13 and MMP-16 were undetectable and MMP-4, MMP-5, and MMP-6 do not exist. Data for MMP-19 to MMP-28 are given in Figure E1 and data for ADAMs in Figure E2: no significant differences were demonstrated in either group. Mean values ± SE are given for three donors infected in separate experiments. Data were analyzed by two-way analysis of variance, followed by Tukey's multiple comparison. Significant effects of a treatment compared with control are indicated. *p < 0.05; **p < 0.01.

 
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.



View larger version (35K):
[in this window]
[in a new window]
  		Figure 2. MTb drives MMP-1 and MMP-7 gene expression and secretion in macrophages. (A) PCR cycle thresholds. The mean cycle threshold (CT) at which PCR amplification enters the logarithmic phase is shown for the 24-h RNA samples analyzed in Figure 1. A low CT indicates high mRNA levels. Open rectangles represent low expression through shaded to solid rectangles, representing high expression. Of the MMPs upregulated by MTb, MMP-1 and MMP-7 were the most highly expressed. CT data for MMP-19 to -28 and ADAMs are given in Figure E3. (B) MMP-1, MMP-7, and MMP-9 secretion. Macrophage MMP secretion at 24 h analyzed by casein (MMP-1 and MMP-7) and gelatin (MMP-9) zymography demonstrated MMP-1 secretion upregulated by MTb, MMP-7 secretion upregulated by both LPS and MTb, and high basal MMP-9 secretion unchanged by LPS or MTb stimulation. Samples were diluted 1:50 for gelatin zymography. (C) Kinetics of MMP-1 and MMP-7 secretion. Macrophage cell culture medium harvested at 24, 48, and 72 h was concentrated 10-fold by lyophilization and analyzed by casein zymography. MTb stimulation increased MMP-1 and MMP-7 secretion compared with uninfected macrophages. (D) MTb increased MMP-1 secretion. Macrophage MMP-1 secretion was analyzed by densitometry of casein zymograms. Control macrophage MMP-1 secretion is indicated by the solid line and MTb-induced secretion by the dashed line. Mean values ± SE from triplicate samples are given. *p < 0.05 by Student's t test. (E) MTb increased MMP-7 secretion. Macrophage MMP-7 secretion was analyzed by densitometry of casein zymograms. Symbols are as per D. (F) MMP-1 Western blot. Macrophage cell culture medium was analyzed at 72 h by Western blotting. The proteolytic bands on casein zymography were confirmed as MMP-1. (G) MMP-7 Western blot. Macrophage cell culture medium was taken at 24, 48, and 72 h was concentrated 10-fold by lyophilization. Western analysis confirmed caseinolytic bands to be 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).



View larger version (14K):
[in this window]
[in a new window]
  		Figure 3. Dexamethasone suppresses MTb-induced MMP-1 and MMP-7 gene expression and secretion. Macrophages were preincubated with dexamethasone at either 0.1 or 1 µM for 2 h and then infected with MTb at an MOI of 1. (A) MMP-1 gene expression. Dexamethasone suppressed MMP-1 gene expression at 24 h in infected macrophages to control levels. (B) MMP-7 gene expression. Dexamethasone reduced MMP-7 gene expression at 24 h in infected macrophages toward control levels but did not completely suppress upregulation. (C) MMP-1 secretion. Dexamethasone suppressed MMP-1 secretion by infected macrophages at 72 h to undetectable levels, analyzed by Western blot. (D) MMP-7 secretion. Dexamethasone reduced MMP-7 secretion by infected macrophages at 72 h toward control concentrations but did not completely suppress it on analysis by casein zymography. Mean values ± SE are given for three donors infected in separate experiments for A and B. Representative data of two experiments performed in triplicate are shown for C and D.

 
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).



View larger version (112K):
[in this window]
[in a new window]
  		Figure 4. MMP-1 and MMP-7 tissue expression in patients with active MTb infection. Paraffin-embedded lung biopsies from six patients with culture-proven MTb infection and from six uninfected patients were immunostained for MMP-1 and MMP-7 as described in METHODS. Left: MMP-1. Right: MMP-7. (A and B) Uninfected biopsy. (C and D) Tb biopsy, low magnification. (E and F) Tb biopsy, higher magnification. (G and H) Tb biopsy, secondary antibody alone. Areas of immunoreactivity appear brown against the blue counterstain. Open arrowheads indicate the central area of caseous necrosis; boxed areas indicate the regions magnified in E and F; solid arrowheads indicate Langhans' giant cells. The giant cells are surrounded by macrophages (epithelioid cells). In Tb, macrophages and Langhans' giant cells around the central area of tissue destruction express MMP-1 (C; higher magnification in E). In uninfected patients, only alveolar macrophages express MMP-1 (A). MMP-7 is expressed by macrophages and Langhans' giant cells around the necrosis (D; higher magnification in F), and also more peripherally in the granuloma (D). Again, in uninfected patients only alveolar macrophages express MMP-7, with inconsistent weak staining at most in other cell types (B). Immunostaining with isotype control primary antibodies demonstrated that binding was specific (G and H). Images shown are representative of six biopsies for each group. Scale bars: 100 µm.

 


View larger version (135K):
[in this window]
[in a new window]
  		Figure 5. MMP-1–expressing cells within MTb granuloma are predominantly CD68 positive. Double-labeling immunohistochemistry was performed on biopsies from MTb-infected patients as detailed in METHODS. MMP-1 immunostaining appears red (A) and CD68 staining appears blue (B). Colocalization of MMP-1 and CD68 is demonstrated in macrophages around the central area of necrosis (C, low magnification; D, higher magnification). Scale bars: 40 µm.

 
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-{gamma} (50 ng/ml), tumor necrosis factor (TNF)-{alpha} (20 and 50 ng/ml), or interleukin (IL)-1{beta} (50 ng/ml), concentrations previously shown to stimulate MMP-1 secretion in monocytes (33, 34). TNF-{alpha}, IL-1{beta}, and IFN-{gamma} 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-{alpha} alone (Figures 6A and 6B). IL-1{beta} 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.



View larger version (23K):
[in this window]
[in a new window]
  		Figure 6. MMP-1/MMP-7 regulation is divergent and only MMP-1 is prostaglandin dependent. (A) Cytokine-stimulated MMP-1 and MMP-7 gene expression. Macrophages were infected with MTb or stimulated with IFN-{gamma} (50 ng/ml), tumor necrosis factor (TNF)-{alpha} (50 ng/ml), or interleukin (IL)-1{beta} (50 ng/ml). Gene expression was analyzed at 24 h by RT-PCR and shown as fold change relative to control. MTb was the greatest stimulus to MMP-1 expression, whereas MMP-7 expression was increased by MTb and TNF-{alpha}. Mean values ± SE are given for three donors infected in separate experiments. (B) Cytokine-stimulated MMP-1 and MMP-7 secretion. Macrophages were infected with MTb or stimulated with IFN-{gamma} (50 ng/ml), TNF-{alpha} (20 ng/ml), or IL-1{beta} (50 ng/ml). MMP-1 and MMP-7 secretion was analyzed at 72 h by Western blotting and casein zymography, respectively. Only MTb increased MMP-1 secretion, whereas both MTb and TNF-{alpha} increased MMP-7 secretion. Data are representative of two experiments performed in triplicate. (C) Effect of prostaglandin inhibition on MMP-1 and MMP-7 secretion. Macrophages were preincubated with indomethacin at 0.1, 1, and 10 µM for 2 h and then infected with MTb. MMP-1 and MMP-7 secretion at 72 h was analyzed by Western blotting and casein zymography, respectively. MMP-1 secretion was inhibited in a dose-dependent manner by indomethacin whereas MMP-7 was not. (D) Densitometric analysis of indomethacin inhibition of MMP-1 and MMP-7 secretion. MMP-1 and MMP-7 secretion by macrophages was analyzed at 72 h by densitometry of casein zymograms. Only MMP-1 secretion was indomethacin sensitive. Mean values ± SE from three samples are given and data are representative of two independent experiments performed in triplicate.

 
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).



View larger version (22K):
[in this window]
[in a new window]
  		Figure 7. MTb and bacillus Calmette-Guérin (BCG) differentially stimulate MMP-1 and MMP-7 secretion. Macrophages were infected with MTb or BCG at an MOI of 1. (A) MMP-1 secretion. Casein zymography and densitometric analysis of MMP-1 secretion by macrophages 72 h postinfection. The casein zymogram was incubated in collagenase buffer for 64 h to increase sensitivity, but no MMP-1 secretion by BCG- infected cells was detected. (B) MMP-1 gene expression. mRNA from MTb- and BCG-infected macrophages extracted at 24 h was analyzed by RT-PCR. MTb- infected macrophage MMP-1 mRNA levels were 2.5-fold higher than BCG-infected macrophage MMP-1 mRNA levels. Mean values ± SE from three donors infected in separate experiments are given. (C) MMP-7 secretion. Casein zymography and densitometric analysis of MMP-7 secretion by macrophages 24 h postinfection are shown. MTb and BCG caused similar increases in macrophage-derived MMP-7 secretion. (D) MMP-7 gene expression. mRNA extracted from MTb- and BCG-infected macrophages at 24 h was analyzed by RT-PCR. MTb and BCG upregulate MMP-7 gene expression equivalently. (E) MTb- and BCG-induced prostaglandin E2 production. Cell culture medium was analyzed at 24 h for prostaglandin E2 concentrations by ELISA. MTb causes threefold more prostaglandin E2 secretion than does BCG. Mean values ± SE from three donors infected in separate experiments are given. (F) Effect of dibutyryl cAMP (B2cAMP) on BCG-induced MMP-1 secretion. Macrophages infected with BCG at an MOI of 1 were costimulated with B2cAMP at 0.1 or 1 µM. MMP-1 secretion was analyzed at 72 h by Western blotting. A 0.1 µM concentration of B2cAMP increased MMP-1 secretion. Data are representative of two independent experiments performed in triplicate.

 
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
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
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-{alpha} 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-{alpha} 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-{alpha} 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. Back

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.

Received in original form May 13, 2005; accepted in final form August 26, 2005


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Dye C, Scheele S, Dolin P, Pathania V, Raviglione MC. Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country. WHO Global Surveillance and Monitoring Project [consensus statement]. JAMA 1999;282:677–686.[Abstract/Free Full Text]
  2. Murray JF. A century of tuberculosis. Am J Respir Crit Care Med 2004; 169:1181–1186.[Free Full Text]
  3. Kaplan G, Post FA, Moreira AL, Wainwright H, Kreiswirth BN, Tanverdi M, Mathema B, Ramaswamy SV, Walther G, Steyn LM, et al. Mycobacterium tuberculosis growth at the cavity surface: a microenvironment with failed immunity. Infect Immun 2003;71:7099–7108.[Abstract/Free Full Text]
  4. Mandell GL, Bennett JE, Dolin R, Douglas RG, editors. Mandell, Douglas, and Bennett's principles and practice of infectious diseases. Philadelphia, PA: Churchill Livingstone; 2000.
  5. North RJ, Jung YJ. Immunity to tuberculosis. Annu Rev Immunol 2004; 22:599–623.[CrossRef][Medline]
  6. Orme IM. The immunopathogenesis of tuberculosis: a new working hypothesis. Trends Microbiol 1998;6:94–97.[CrossRef][Medline]
  7. Kaufmann SH. A short history of Robert Koch's fight against tuberculosis: those who do not remember the past are condemned to repeat it. Tuberculosis (Edinb) 2003;83:86–90.
  8. Crystal RG, West JB. The lung: scientific foundations. Philadelphia, PA: Lippincott; 1997.
  9. Owen CA, Campbell EJ. The cell biology of leukocyte-mediated proteolysis. J Leukoc Biol 1999;65:137–150.[Abstract]
  10. D'Armiento J, Dalal SS, Okada Y, Berg RA, Chada K. Collagenase expression in the lungs of transgenic mice causes pulmonary emphysema. Cell 1992;71:955–961.[CrossRef][Medline]
  11. Taraseviciene-Stewart L, Scerbavicius R, Choe KH, Moore M, Sullivan A, Nicolls MR, Fontenot AP, Tuder RM, Voelkel NF. An animal model of autoimmune emphysema. Am J Respir Crit Care Med 2005;171: 734–742.[Abstract/Free Full Text]
  12. Churg A, Wang RD, Tai H, Wang X, Xie C, Wright JL. Tumor necrosis factor-{alpha} drives 70% of cigarette smoke-induced emphysema in the mouse. Am J Respir Crit Care Med 2004;170:492–498.[Abstract/Free Full Text]
  13. Choe KH, Taraseviciene-Stewart L, Scerbavicius R, Gera L, Tuder RM, Voelkel NF. Methylprednisolone causes matrix metalloproteinase- dependent emphysema in adult rats. Am J Respir Crit Care Med 2003; 167:1516–1521.[Abstract/Free Full Text]
  14. Brinckerhoff CE, Matrisian LM. Matrix metalloproteinases: a tail of a frog that became a prince. Nat Rev Mol Cell Biol 2002;3:207–214.[CrossRef][Medline]
  15. McCawley LJ, Matrisian LM. Matrix metalloproteinases: they're not just for matrix anymore! Curr Opin Cell Biol 2001;13:534–540.[CrossRef][Medline]
  16. Filippov S, Caras I, Murray R, Matrisian LM, Chapman HA Jr, Shapiro S, Weiss SJ. Matrilysin-dependent elastolysis by human macrophages. J Exp Med 2003;198:925–935.[Abstract/Free Full Text]
  17. Parks WC, Wilson CL, Lopez-Boado YS. Matrix metalloproteinases as modulators of inflammation and innate immunity. Nat Rev Immunol 2004;4:617–629.[CrossRef][Medline]
  18. Taggart CC, Greene CM, Carroll TP, O'Neill SJ, McElvaney NG. Elastolytic proteases: inflammation resolution and dysregulation in chronic infective lung disease. Am J Respir Crit Care Med 2005;171:1070–1076.[Free Full Text]
  19. Campbell EJ, Cury JD, Shapiro SD, Goldberg GI, Welgus HG. Neutral proteinases of human mononuclear phagocytes: cellular differentiation markedly alters cell phenotype for serine proteinases, metalloproteinases, and tissue inhibitor of metalloproteinases. J Immunol 1991;146: 1286–1293.[Abstract]
  20. Welgus HG, Campbell EJ, Cury JD, Eisen AZ, Senior RM, Wilhelm SM, Goldberg GI. Neutral metalloproteinases produced by human mononuclear phagocytes: enzyme profile, regulation, and expression during cellular development. J Clin Invest 1990;86:1496–1502.
  21. Lai WC, Zhou M, Shankavaram U, Peng G, Wahl LM. Differential regulation of lipopolysaccharide-induced monocyte matrix metalloproteinase (MMP)-1 and MMP-9 by p38 and extracellular signal- regulated kinase 1/2 mitogen-activated protein kinases. J Immunol 2003;170:6244–6249.[Abstract/Free Full Text]
  22. Shapiro SD, Kobayashi DK, Pentland AP, Welgus HG. Induction of macrophage metalloproteinases by extracellular matrix: evidence for enzyme- and substrate-specific responses involving prostaglandin- dependent mechanisms. J Biol Chem 1993;268:8170–8175.[Abstract/Free Full Text]
  23. Busiek DF, Baragi V, Nehring LC, Parks WC, Welgus HG. Matrilysin expression by human mononuclear phagocytes and its regulation by cytokines and hormones. J Immunol 1995;154:6484–6491.[Abstract]
  24. Goetzl EJ, Banda MJ, Leppert D. Matrix metalloproteinases in immunity. J Immunol 1996;156:1–4.[Abstract]
  25. Flynn JL, Chan J. Immunology of tuberculosis. Annu Rev Immunol 2001;19:93–129.[CrossRef][Medline]
  26. Munger JS, Chapman HA. Tissue destruction by proteases. In: Rom WN, Garay SM, editors. Tuberculosis, 1st ed. Boston: Little, Brown and Company; 1996. pp. 353–361.
  27. Elkington PTG, Nutall RK, Boyle JJ, Edwards DR, Friedland JS. Mycobacterium tuberculosis (MTb) stimulates matrix metalloproteinase-1 and -7 expression in human monocytic cells regulated by divergent mechanisms [abstract]. J Leukoc Biol 2004;40.
  28. Elkington PTG, Nutall RK, O'Kane CM, Boyle JJ, Edwards DR, Friedland JS. Mechanisms regulating matrix metalloproteinase gene expression and secretion in pulmonary tuberculosis [abstract]. Thorax 2004;59:T5.
  29. Nuttall RK, Pennington CJ, Taplin J, Wheal A, Yong VW, Forsyth PA, Edwards DR. Elevated membrane-type matrix metalloproteinases in gliomas revealed by profiling proteases and inhibitors in human cancer cells. Mol Cancer Res 2003;1:333–345.[Abstract/Free Full Text]
  30. Leber TM, Balkwill FR. Zymography: a single-step staining method for quantitation of proteolytic activity on substrate gels. Anal Biochem 1997;249:24–28.[CrossRef][Medline]
  31. Joint Tuberculosis Committee of the British Thoracic Society. Chemotherapy and management of tuberculosis in the UK: recommendations 1998. Thorax 1998;53:536–548.[Abstract/Free Full Text]
  32. Shapiro SD, Campbell EJ, Kobayashi DK, Welgus HG. Dexamethasone selectively modulates basal and lipopolysaccharide-induced metalloproteinase and tissue inhibitor of metalloproteinase production by human alveolar macrophages. J Immunol 1991;146:2724–2729.[Abstract]
  33. Zhang Y, McCluskey K, Fujii K, Wahl LM. Differential regulation of monocyte matrix metalloproteinase and TIMP-1 production by TNF-{alpha}, granulocyte-macrophage CSF, and IL-1{beta} through prostaglandin-dependent and -independent mechanisms. J Immunol 1998;161: 3071–3076.[Abstract/Free Full Text]
  34. Zhou M, Zhang Y, Ardans JA, Wahl LM. Interferon-{gamma} differentially regulates monocyte matrix metalloproteinase-1 and -9 through tumor necrosis factor-{alpha} and caspase 8. J Biol Chem. 2003.
  35. Wickremasinghe MI, Thomas LH, O'Kane CM, Uddin J, Friedland JS. Transcriptional mechanisms regulating alveolar epithelial cell-specific CCL5 secretion in pulmonary tuberculosis. J Biol Chem 2004;279: 27199–27210.[Abstract/Free Full Text]
  36. Newport MJ, Huxley CM, Huston S, Hawrylowicz CM, Oostra BA, Williamson R, Levin M. A mutation in the interferon-{gamma}-receptor gene and susceptibility to mycobacterial infection. N Engl J Med 1996;335: 1941–1949.[Abstract/Free Full Text]
  37. Lopez-Maderuelo D, Arnalich F, Serantes R, Gonzalez A, Codoceo R, Madero R, Vazquez JJ, Montiel C. Interferon-{gamma} and interleukin-10 gene polymorphisms in pulmonary tuberculosis. Am J Respir Crit Care Med 2003;167:970–975.[Abstract/Free Full Text]
  38. Wahl LM, Olsen CE, Sandberg AL, Mergenhagen SE. Prostaglandin regulation of macrophage collagenase production. Proc Natl Acad Sci USA 1977;74:4955–4958.[Abstract/Free Full Text]
  39. Corcoran ML, Stetler-Stevenson WG, Brown PD, Wahl LM. Interleukin 4 inhibition of prostaglandin E2 synthesis blocks interstitial collagenase and 92-kDa type IV collagenase/gelatinase production by human monocytes. J Biol Chem 1992;267:515–519.[Abstract/Free Full Text]
  40. Welgus HG, Jeffrey JJ, Stricklin GP, Roswit WT, Eisen AZ. Characteristics of the action of human skin fibroblast collagenase on fibrillar collagen. J Biol Chem 1980;255:6806–6813.[Free Full Text]
  41. Davidson JM. Biochemistry and turnover of lung interstitium. Eur Respir J 1990;3:1048–1063.[Abstract]
  42. Shapiro SD, Endicott SK, Province MA, Pierce JA, Campbell EJ. Marked longevity of human lung parenchymal elastic fibers deduced from prevalence of D-aspartate and nuclear weapons-related radiocarbon. J Clin Invest 1991;87:1828–1834.
  43. Shapiro SD, Campbell EJ, Welgus HG, Senior RM. Elastin degradation by mononuclear phagocytes. Ann N Y Acad Sci 1991;624:69–80.[Medline]
  44. Imai K, Dalal SS, Chen ES, Downey R, Schulman LL, Ginsburg M, D'Armiento J. Human collagenase (matrix metalloproteinase-1) expression in the lungs of patients with emphysema. Am J Respir Crit Care Med 2001;163:786–791.[Abstract/Free Full Text]
  45. Finlay GA, O'Driscoll LR, Russell KJ, D'Arcy EM, Masterson JB, FitzGerald MX, O'Connor CM. Matrix metalloproteinase expression and production by alveolar macrophages in emphysema. Am J Respir Crit Care Med 1997;156:240–247.[Abstract/Free Full Text]
  46. Segura-Valdez L, Pardo A, Gaxiola M, Uhal BD, Becerril C, Selman M. Upregulation of gelatinases A and B, collagenases 1 and 2, and increased parenchymal cell death in COPD. Chest 2000;117:684–694.[Abstract/Free Full Text]
  47. Selman M, Montano M, Ramos C, Vanda B, Becerril C, Delgado J, Sansores R, Barrios R, Pardo A. Tobacco smoke-induced lung emphysema in guinea pigs is associated with increased interstitial collagenase. Am J Physiol 1996;271:L734–L743.
  48. Balbin M, Fueyo A, Knauper V, Lopez JM, Alvarez J, Sanchez LM, Quesada V, Bordallo J, Murphy G, Lopez-Otin C. Identification and enzymatic characterization of two diverging murine counterparts of human interstitial collagenase (MMP-1) expressed at sites of embryo implantation. J Biol Chem 2001;276:10253–10262.[Abstract/Free Full Text]
  49. Wahl SM, Wahl LM, McCarthy JB, Chedid L, Mergenhagen SE. Macrophage activation by mycobacterial water soluble compounds and synthetic muramyl dipeptide. J Immunol 1979;122:2226–2231.[Abstract/Free Full Text]
  50. Chang JC, Wysocki A, Tchou-Wong KM, Moskowitz N, Zhang Y, Rom WN. Effect of Mycobacterium tuberculosis and its components on macrophages and the release of matrix metalloproteinases. Thorax 1996;51:306–311.[Abstract/Free Full Text]
  51. Price NM, Farrar J, Tran TT, Nguyen TH, Tran TH, Friedland JS. Identification of a matrix-degrading phenotype in human tuberculosis in vitro and in vivo. J Immunol 2001;166:4223–4230.[Abstract/Free Full Text]
  52. Rivera-Marrero CA, Schuyler W, Roser S, Ritzenthaler JD, Newburn SA, Roman J. M. tuberculosis induction of matrix metalloproteinase-9: the role of mannose and receptor-mediated mechanisms. Am J Physiol Lung Cell Mol Physiol 2002;282:L546–L555.[Abstract/Free Full Text]
  53. Quiding-Jarbrink M, Smith DA, Bancroft GJ. Production of matrix metalloproteinases in response to mycobacterial infection. Infect Immun 2001;69:5661–5670.[Abstract/Free Full Text]
  54. Price NM, Gilman RH, Uddin J, Recavarren S, Friedland JS. Unopposed matrix metalloproteinase-9 expression in human tuberculous granuloma and the role of TNF-{alpha}-dependent monocyte networks. J Immunol 2003;171:5579–5586.[Abstract/Free Full Text]
  55. Pierce RA, Sandefur S, Doyle GA, Welgus HG. Monocytic cell type-specific transcriptional induction of collagenase. J Clin Invest 1996;97: 1890–1899.[Medline]
  56. Grosset J. Mycobacterium tuberculosis in the extracellular compartment: an underestimated adversary. Antimicrob Agents Chemother 2003;47: 833–836.[Free Full Text]
  57. Turner OC, Basaraba RJ, Orme IM. Immunopathogenesis of pulmonary granulomas in the guinea pig after infection with Mycobacterium tuberculosis. Infect Immun 2003;71:864–871.[Abstract/Free Full Text]
  58. Thwaites GE, Nguyen DB, Nguyen HD, Hoang TQ, Do TT, Nguyen TC, Nguyen QH, Nguyen TT, Nguyen NH, Nguyen TN, et al. Dexamethasone for the treatment of tuberculous meningitis in adolescents and adults. N Engl J Med 2004;351:1741–1751.[Abstract/Free Full Text]
  59. Jonat C, Rahmsdorf HJ, Park KK, Cato AC, Gebel S, Ponta H, Herrlich P. Antitumor promotion and antiinflammation: down-modulation of AP-1 (Fos/Jun) activity by glucocorticoid hormone. Cell 1990;62:1189–1204.[CrossRef][Medline]
  60. Saren P, Welgus HG, Kovanen PT. TNF-{alpha} and IL-1{beta} selectively induce expression of 92-kDa gelatinase by human macrophages. J Immunol 1996;157:4159–4165.[Abstract]
  61. Mahairas GG, Sabo PJ, Hickey MJ, Singh DC, Stover CK. Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis. J Bacteriol 1996;178:1274–1282.[Abstract/Free Full Text]
  62. Tse HM, Josephy SI, Chan ED, Fouts D, Cooper AM. Activation of the mitogen-activated protein kinase signaling pathway is instrumental in determining the ability of Mycobacterium avium to grow in murine macrophages. J Immunol 2002;168:825–833.[Abstract/Free Full Text]
  63. Harris SG, Padilla J, Koumas L, Ray D, Phipps RP. Prostaglandins as modulators of immunity. Trends Immunol 2002;23:144–150.[CrossRef][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Online Supplement
Right arrow All Versions of this Article:
200505-753OCv1
172/12/1596    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Elkington, P. T. G.
Right arrow Articles by Friedland, J. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Elkington, P. T. G.
Right arrow Articles by Friedland, J. S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Cell Mol. Biol.
Copyright © 2005 American Thoracic Society