Address of Corresponding Author

Respiration 2005;72:166-175 (DOI: 10.1159/000084048)

 Outline

• Key Words
• Abstract
• Introduction
• Materials and Methods
• Study Group
• Specimen Collection and Processing
• Enzyme Immunoassay
• Gelatin Zymography
• Reverse-Transcriptase Polymerase Chain Reaction Analysis
• Immunohistochemistry
• Statistical Analysis
• Results
• MMP-9 and TIMP-1 in Pleural Fluid
• Gelatin Zymography in Pleural Effusions
• Reverse-Transcriptase Polymerase Chain Reaction
• Immunohistochemistry
• Discussion
• References

• Author Contacts
• Article Information
• Publication Details
• Drug Dosage / Copyright

  Key Words

• Matrix metalloproteinase-9
• Tissue inhibitors of matrix metalloproteinase-1
• Tuberculous pleural effusion
• Malignant pleural effusion
• Lung cancer

  Abstract

Background: Matrix metalloproteinase (MMP)-9 has been implicated in the development of pleural effusions. Objectives: The aim of this study was to assess the expression of MMP-9 in pleural effusions of tuberculosis, lung cancer and transudates. Methods: Ninety-one patients (37 tuberculous pleural effusions, 42 malignant pleural effusions of lung cancer and 12 transudates) were included. Concentrations of pleural fluid MMP-9 and tissue inhibitors of matrix metalloproteinase (TIMP)-1 were determined by immunoassay. We also investigated the cellular localization of MMP-9 and TIMP-1 by reverse-transcriptase polymerase chain reaction on lymphocytes from pleural effusions and by immunohistochemical analysis of pleural tissues. Results: Pleural fluid MMP-9 levels, MMP-9/total protein and MMP-9/TIMP-1 ratios were significantly higher in tuberculous pleural effusions, whilst TIMP-1 levels were similar in the three groups. MMP-9 levels positively correlated with TIMP-1 and lactate dehydrogenase levels, and negatively with pH and glucose levels in pleural effusions. MMP-9 mRNA expression in lymphocytes tended to be higher in malignant pleural effusions of lung cancer than in the other groups without reaching statistical significance. The strongest immunoreactivity for MMP-9 was observed in epithelioid cells of tuberculous pleural tissues. Much lower levels of MMP-9 expression were found in tumor cells of pleural tissues. Conclusions: MMP-9 is increased in tuberculous pleural effusions compared with transudates and malignant pleural effusions of lung cancer and is produced predominantly by epithelioid cells in the granulomas of tuberculous pleural tissues.

Copyright © 2005 S. Karger AG, Basel

 Introduction

The proteolytic balance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) is important not only in normal tissue remodeling, but also in various pathological conditions [1, 2, 3, 4]. Proteolytic processes may play a role in the formation of pleural effusions by increasing vascular permeability, and therefore by facilitating fluid influx into the pleural space [5]. The presence and enzymatic activities of MMPs and TIMPs have been identified in pleural effusions [6, 7].

Tissue damage is a characteristic manifestation of infection by Mycobacterium tuberculosis (Mtb). Proteolysis by macrophage-secreted proteases has been implicated in such destructive processes [8, 9]. In this regard, the proteolytic action of MMPs may be involved in the pathogenesis of tuberculosis, like many other diseases associated with tissue destruction. Several studies have reported that macrophages and monocytes release MMP-9 in response to Mtb or its cellular components [10, 11, 12]. An in vivo study demonstrated the activation of MMP-2 and MMP-9 in the lungs of mice infected with Mtb [13]. One study evaluated MMPs and TIMPs in tuberculous pleural effusions, and found that immunoreactive pleural fluid concentrations of MMPs were higher in 21 patients with tuberculosis when compared with patients with congestive heart failure [14].

Lung cancer is another common cause of lymphocyte-predominant pleural effusions as observed in tuberculous pleural effusions. MMPs have been known to play a role in the pathogenesis of malignancy [1, 15, 16]. They have been reported to be expressed in lung cancer tissues and to be elevated in the sera and pleural effusions of lung cancer patients [17, 18, 19]. However, direct comparisons of MMPs and TIMPs between tuberculous pleural effusions and malignant pleural effusions of lung cancer have not been reported. Furthermore, their specific cellular expression and localization in pleural diseases have not been assessed in detail. Although pleural mesothelial cells have been proposed as a source of MMP-9 [7, 20], many other cell types such as macrophages, monocytes, neutrophils, stromal cells and tumor cells can produce MMP-9 [1, 21]. Lymphocytes, the most predominant cell type in the pleural effusions of both tuberculosis and lung cancer, may also be sources of MMP-9 [22]. The relative responsibility of each cell for the production of MMP-9 may vary according to the underlying diseases.

In this study, we determined the concentrations of MMP-9 and TIMP-1 in tuberculous pleural effusions compared with malignant pleural effusions of lung cancer and transudates. We also assessed their expression in cells from pleural effusions and pleural tissues.

 Materials and Methods

 Study Group

A total of 91 patients with pleural effusions were enrolled from January 2002 to March 2003. Thirty-seven patients had tuberculous pleural effusions (mean age 37.2 ± 17.2 years; 21 males and 16 females). Forty-two patients had malignant pleural effusions due to lung cancer (mean age 61.7 ± 11.4 years; 23 males and 19 females). Cell types of lung cancer were adenocarcinoma (n = 28), squamous cell carcinoma (n = 8), large cell carcinoma (n = 2) and small cell carcinoma (n = 4). Twelve patients had transudative effusions (mean age 66.3 ± 16.5 years; 7 males and 5 females). Underlying diseases causing transudates were congestive heart failure (n = 10) and chronic renal failure (n = 2).

Pleural fluid was designated as exudates or transudates according to Light's criteria [23, 24]. All pleural effusions had definite etiologies documented by the examination of effusion biochemistry, cytology, tissue biopsies, acid-fast staining and clinical follow-up. The diagnostic criteriaused for the tuberculous pleural effusion and the malignant effusion were described previously [25, 26]. Patients were diagnosed as having tuberculosis effusion when they met any of the following criteria: Mtb isolated from pleural fluid or pleural tissue (n = 4); granulomas in the pleural tissue showing staining for acid-fast bacilli (n = 17); granulomas in pleural tissue that did not stain for acid-fast bacilli, but showed a response to antituberculous treatment (n = 13), or a positive sputum culture finding for Mtb in the presence of a pleural effusion (n = 3). Malignant pleural effusions were diagnosed either by a positive pleural fluid cytologic result or by the presence of malignant cells, as identified in a pleural biopsy specimen. Pleural fluid protein levels were 16.5 mg/ml (range 13.5-21.0) in transudates, 50.0 mg/ml (46.0-54.0) in tuberculous pleural effusions and 44.0 mg/ml (37.0-49.0) in malignant pleural effusions of lung cancer. Pleural fluid lymphocyte counts were 582/µl (range 452-847) in transudates, 2,587/µl (1,711-3,445) in tuberculous pleural effusions and 2,071/µl (1,012-3,088) in malignant pleural effusions of lung cancer. The protocol was approved by the hospital's Ethical Committee, and written consent was obtained from the patients.

 Specimen Collection and Processing

Thoracentesis was performed in the usual manner, and the pleural tissue samples were obtained by blind biopsies with Abram's needle, except four, which were biopsied by thoracoscopy. A portion of the pleural effusion sample was submitted for acid-fast staining, cytologic examination and the measurement of pH, protein, albumin, lactate dehydrogenase (LDH) and glucose. Total cell, white cell and differential cell counts (Giemsa stain) were obtained by counting at least 200 cells under a light microscope. Another portion of sample was centrifuged at 3,000 g per minute for 15 min, and the supernatants were frozen at -70°C. Lymphocytes were extracted from the cell pellet by dextran sedimentation followed by Ficoll-Plaque density gradient centrifugation [27].

 Enzyme Immunoassay

The immunoreactive levels of MMP-9 and TIMP-1 were determined in the supernatants of pleural effusions utilizing sandwich enzyme immunoassays (R&D Systems, Minneapolis, Minn., USA).

 Gelatin Zymography

Gelatin zymography was performed by using 8% SDS-polyacrylamide gel containing 1 mg/ml gelatin, as previously described [28]. In brief, each sample was diluted 1:10, and 5-µl aliquots were mixed with sample buffer and electrophoresed on gels at a constant 100 V for 2 h. The gels were then incubated for 1 h at room temperature in 2.5% Triton X-100, followed by an overnight incubation at 37°C in gelatinase substrate buffer (50 mM Tris, 200 mM NaCl, 5 mM CaCl₂ and 0.2% Brij35, pH 7.5). Bands of enzymatic activity were visualized by negative staining with standard Coomassie brilliant blue dye solution followed by subsequent destaining with 50% methanol. The gels were dried on cellophane, and signals from the stained bands were detected and quantified using an Image Analyzer (BioRad, Calif., USA).

 Reverse-Transcriptase Polymerase Chain Reaction Analysis

Total RNA was extracted by lysing lymphocytes with Trizol Reagent (Gibco BRL Life Technologies, Rockville, Md., USA), according to the manufacturer's instructions. RNA integrity was confirmed by electrophoresis in 1.5% agarose gel and ethidium bromide staining. Total cellular RNA (2 µg) was used for the complementary DNA (cDNA) synthesis. The manufacturer's protocol was followed using the supplied oligo(dT) primers and Moloney murine leukemia virus reverse transcriptase in a reaction volume of 20 µl (Superscript Preamplification System, Gibco BRL Life Technologies). Sequences were amplified from cDNA by a polymerase chain reaction (PCR) using specific primers for MMP-9 [29], TIMP-1 [30] and -actin, which were obtained from published sequences, and synthesized commercially (Bioneer, Daejon, South Korea). The specific primers used were as follows: MMP-9 sense primer 5'-GGG GAA GAT GCT GCT GTT CA-3', antisense primer 5'-GGT CCC AGT GGG GAT TTA CA-3'; TIMP-1 sense primer 5'-GGG GAC ACC AGA AGT CAA CCA GA-3', antisense primer 5'-CTT TTC AGA GCC TTG GAG GAG CT-3'; -actin sense primer 5'-GAC CTG ACT GAC TAC CTC AT-3', antisense primer 5'-TCG TCA TAC TCC TGC TTG CT-3'. The expected amplification product sizes were 308, 400 and 542 base pairs (bp), respectively. PCR amplifications were performed in 20 µl containing 2 µl of 10× PCR buffer, 1 µl of the synthesized cDNA sample, 1 µl of 20 pmol each oligonucleotide primer, 2.5 mM MgCl₂, 0.2 mM dNTPs mixture, and 1 unit of Taq polymerase (AmpliTaq Gold, Perkin Elmer, Roche, N.J., USA). Initial template denaturation at 95°C for 5 min was followed by 24 or 30 cycles at 95°C for 1 min, 58 or 62°C for 1 min, and 72°C for 1 min, and this was followed with a final extension step of 10 min at 72°C. For MMP-9 and TIMP-1, the annealing temperature was 62°C, and for -actin 58°C. The number of cycles carried out for MMP-9 and TIMP-1 was 30, and for -actin 24. Each PCR product (5 µl) was electrophoresed on a 1.5% agarose gel containing ethidium bromide, and a DNA molecular weight marker (100-bp ladder) was used for comparison. Signals from the stained bands were detected and quantified using an Image Analyzer (BioRad). To allow semiquantification of mRNA levels, MMP-9 and TIMP-1 expression was standardized to -actin expression.

 Immunohistochemistry

Paraffin tissue blocks were sectioned at 4 µm and stained with hematoxylin-eosin. Immunohistochemical studies were carried out on deparaffinized sections using the streptavidin-biotin-peroxidase method (Cap-Plus^TM Detection Kit, Zymed Laboratories, San Francisco, Calif., USA). Briefly, the sections were first treated with a blocking protein to minimize the nonspecific binding and then incubated overnight at 4°C with mouse monoclonal antihuman MMP-9 (Neomarker, Fremont, Calif., USA) or rabbit antihuman anti- TIMP-1 (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) antibody, as primary antibody, then with biotinylated secondary antibody solution for 13 min, and finally with streptavidin for 13 min. Peroxidase activity was visualized using 3,3'-diamino-benzidine and counterstained with hematoxylin. As controls, sections were incubated with nonimmunized mouse Ig (IgG), instead of specific monoclonal antibodies, and then processed according to the above procedure. The distributions and intensities of the immunohistochemical positive reactions were evaluated by three independent observers blind to the tissues and antibodies used. The staining intensity was scored semiquantitatively as negative, weak, moderate or strong; in heterogeneous cases, the rating of the predominant pattern prevailed. For the rare cases with discordant scoring between the observers, a final score was reached by consensus.

 Statistical Analysis

Demographic data were expressed as means ± SD. Since MMP-9 and TIMP-1 concentrations were not normally distributed, we expressed each concentration as the median, 25th and 75th quartiles. Comparison among groups was done using analysis of variance (ANOVA) on ranks (Kruskal-Wallis) with post-hoc pairwise testing (Dunn's method). Correlations were analyzed with the Spearman rank order correlation. Differences were regarded as statistically significant at p < 0.05.

 Results

 MMP-9 and TIMP-1 in Pleural Fluid

Pleural fluid concentrations of MMP-9 were significantly increased in lung cancer (median 8.8, interquartile range 5.2-32.6 ng/ml) and even higher in tuberculosis (35.3, 18.1-71.6 ng/ml) compared with transudates (2.8, 1.5-6.2 ng/ml; p < 0.05 and p < 0.001, respectively). Pleural fluid concentrations of MMP-9 were significantly higher in tuberculosis than in lung cancer (p < 0.001; fig. 1A). Since protein levels in transudates were too low compared with the other groups, we performed intergroup comparisons using levels adjusted with respect to pleural fluid protein concentrations, as well as with absolute values. When MMP-9 levels were adjusted with respect to pleural fluid total protein concentrations, the ratios [(MMP-9/total protein) × 10^-6] were significantly higher in tuberculosis (7.3, 3.8-14.3) than in lung cancer (1.9, 1.1-7.4) and in transudates (1.6, 1.2-2.9; both, p < 0.001; fig. 1C). Pleural fluid concentrations of TIMP-1 were significantly higher in lung cancer (2,066.9, 1,497.5-2,843.6 ng/ml) and tuberculosis (2,202.7, 1,940.0-3,069.2 ng/ml) than in transudates (874.3, 817.3-1,159.3 ng/ml; both, p < 0.001; fig. 1B). However, when adjusted with respect to pleural fluid protein concentrations, the ratios [(TIMP-1/total protein) × 10^-4] were not different among the three groups (tuberculosis: 4.2, 3.8-6.7; lung cancer: 5.0, 3.0-6.5; transudates: 5.3, 4.1-8.0; fig. 1D). Pleural fluid MMP-9/TIMP-1 ratios (×10^-2) were therefore significantly higher in tuberculosis (1.3, 0.8-3.0) than in lung cancer (0.6, 0.3-1.3) and in transudates (0.3, 0.2-0.5; both, p < 0.001). Pleural fluid MMP-9 levels positively correlated with TIMP-1 levels (r = 0.53, p < 0.001) and LDH levels (r = 0.40, p < 0.001), and negatively with pH (r = -0.42, p < 0.001) and glucose levels (r = -0.44, p < 0.001) in pleural effusions of all patients (fig. 2). Similar significant correlations were found when the groups were analyzed separately (data not shown). There was no significant relationship between pleural fluid MMP-9 levels and total cell count or differential cell profiles.

Fig. 1. MMP-9 concentrations (A), TIMP-1 concentrations (B), MMP-9/total protein (C) and TIMP-1/total protein (D) in pleural effusions of transudates (TR), tuberculosis (TB) and lung cancer (LC). Horizontal lines represent median values. p values of significant differences are shown.

Fig. 2. Correlations between MMP-9 levels and parameters in pleural effusions of all patients: TIMP-1 levels (A), LDH levels (B), pH (C) and glucose levels (D) in pleural effusions are depicted.

 Gelatin Zymography in Pleural Effusions

Consistent with the results obtained from the enzyme immunoassay, the activities of the 92-kDa proform of MMP-9 (pMMP-9) were significantly increased in tuberculous pleural effusions compared with malignant pleural effusions of lung cancer and transudates (p < 0.01). The activities of the 72-kDa proform of MMP-2 (pMMP-2) were generally high, and showed no significant differences among the three groups. The activities of the 67-kDa active form of MMP-2 (aMMP-2) were very low, and showed no significant differences among the three groups (fig. 3).

Fig. 3. Identification of gelatinolytic activities in pleural effusions of transudates (TR), tuberculosis (TB) and lung cancer (LC). Gelatin zymography was performed (A), and densitometry scans of the bands were obtained. Five representative samples are shown for each group. Bar graphs illustrate the relative ratio of pMMP-9 to pMMP-2 densities, represented as mean ± SEM (B). * p < 0.05 compared with LC and TR.

 Reverse-Transcriptase Polymerase Chain Reaction

Although MMP-9 mRNA expression in lymphocytes tended to be higher in malignant pleural effusions of lung cancer than in the other groups,the differences did not reach statistical significance (p = 0.085). TIMP-1 mRNA expression was similar among the three groups (fig. 4).

Fig. 4. Semiquantitative RT-PCR analysis of MMP-9 and TIMP-1 in lymphocytes obtained from pleural effusions of transudates (TR), tuberculosis (TB) and lung cancer (LC) was performed in three groups (A), and a densitometry scan of the bands was obtained. Five representative samples are shown for each group. Bar graphs illustrate relative ratios of MMP-9 (B) and TIMP-1 (C) to -actin mRNA levels, represented as means ± SEM.

 Immunohistochemistry

In tuberculous pleural tissues, immunoreactivity for MMP-9 was most prominent in epithelioid cells within granulomas, showing strong staining in all cases. Positive staining was also observed in mesothelial cells at weak levels. In pleural tissues of metastatic lung cancer, weak to moderate immunoreactivity for MMP-9 was present in the tumor cells (fig. 5). Similarly, in both tuberculous pleural tissues and pleural tissues of metastatic lung cancer, MMP-9 expression was generally weak in lymphocytes, and negative in other cells including endothelial cells and stromal cells. TIMP-1 was also expressed predominantly in epithelioid cells in tuberculous pleural tissues, and in tumor cells in pleural tissues of metastatic lung cancer. TIMP-1 was expressed by many more cell types in the pleural cavity than was MMP-9 (fig. 6).

Fig. 5. Immunohistochemical analysis for MMP-9 in pleural tissues of tuberculosis and lung cancer. A Immunoreactivity in the granuloma shows marked staining contrasting with negative to faint signals of surrounding inflammatory and stromal cells in tuberculous pleural tissues. Original magnification ×100. B Strong staining of epithelioid cells is present in the granuloma of tuberculous pleuritis. The inset shows weak staining of the mesothelial cells in the same specimen. Original magnification ×400. Low levels of immunoreactivity are observed in metastatic cells of adenocarcinoma (C), squamous cell carcinoma (D) and small cell carcinoma (E) in pleural tissues of lung cancer. C-E Original magnifications ×400.

Fig. 6. Immunohistochemical analysis for TIMP-1 in pleural tissues of tuberculosis and lung cancer. A Immunoreactivity in epithelioid cells in the granuloma is noted with less intense staining of lymphocytes and fibroblasts in tuberculous pleural tissues. Original magnification ×400. Immunoreactivity is observed in metastatic cells of adenocarcinoma (B), squamous cell carcinoma (C) and small cell carcinoma (D) in pleural tissues of lung cancer. B-D Original magnifications ×400.

 Discussion

MMPs, together with TIMPs, have been suggested to play a role in the pathogenesis of pleural effusions, as in many other disease processes [6, 7, 31]. There have been a few reports concerning MMPs and TIMPs in pleural effusions, one of which reported higher concentrations of MMP-9 in tuberculous pleural effusions than in transudates [14]. In the present study, tuberculous pleural effusions showed higher concentrations and activities of MMP-9 than not only the transudates, but also the malignant pleural effusions of lung cancer.

Regarding the cellular sources for MMPs, previous studies concentrated particularly on the actions of mesothelial cells as responsible for the release of MMPs into pleural effusions [6, 20]. In our analysis of cells from the pleural effusions, we first focused on lymphocytes to evaluate MMP-9 and TIMP-1 mRNA expression, since they are the most predominant cells in pleural effusions of both lung cancer and tuberculosis. Lymphocytes from pleural effusions expressed MMP-9 mRNA, and its expression tended to be higher in malignant pleural effusions of lung cancer. This finding indicates that lymphocytes may serve as a source of MMP-9 in pleural effusions. However, MMP-9 immunoreactivity in lymphocytes was much weaker compared with epithelioid cells in tuberculous pleural tissues. In addition, pleural fluid MMP-9 levels were not found to correlate with the number of lymphocytes in pleural effusions. From these results, lymphocytes do not appear to be a predominant source for MMP-9 in tuberculous pleural effusions.

In pulmonary tuberculosis, macrophages play a key role in the pathogenetic process by releasing many kinds of proteases and cytokines, and by introducing a protective cellular immune response [32, 33, 34]. Proteases, secreted mainly by macrophages, have been reported to cause the liquefaction of surrounding tissues and initiate cavity formation [8, 9, 35]. MMP-9, as one of these proteases, is also known to be released from macrophages and has been implicated to be involved in the pathogenesis of pulmonary tuberculosis [12, 36]. We previously noted prominently higher MMP-9 mRNA expression in the alveolar macrophages from bronchoalveolar lavage of active pulmonary tuberculosis than those of healthy controls [unpubl. report]. In this study, we observed that epithelioid cells consistently expressed MMP-9 in all of the tuberculous pleural tissues examined, and that the intensity of its staining was much stronger than any other cells, including lymphocytes and mesothelial cells. Taken together, these results suggest a significant role of cells from the monocyte/macrophage lineage in the production of MMP-9 in tuberculous pleural effusions, as is the case in pulmonary tuberculosis.

An intriguing finding is the significant correlation between LDH and MMP-9 levels. The levels of LDH in the pleural fluid reflect pleural inflammation [37]. Several studies have reported on the relationship between MMP-9 and the inflammatory process. Inflammatory cytokines such as TNF- and IL-1 have been shown to induce MMP-9 secretion by macrophages [38], and conversely, MMPs can induce the release of proinflammatory cytokines [39, 40]. In addition, MMPs are required for leukocyte migration to sites of infection [21], such as occurs during cell influx in antimycobacterial granuloma formation. MMP-9 levels negatively correlated with pH and glucose levels in pleural effusions. The pathogenetic mechanisms and clinical implications of pleural pH and glucose level changes have been described previously, although not specifically addressed in the context of tuberculous pleural effusions. In parapneumonic effusions, low pH and glucose content have been known to be related to the fibropurulent process, which leads to complicated parapneumonic effusions and empyema [37, 41]. The inverse correlation between MMP-9 levels and either pH or glucose levels may suggest that MMP-9 is either directly or indirectly involved in the fibropurulent reactions in pleural effusions. In malignant pleural effusions, decreases in pH and glucose levels were reported to be caused mainly by impaired glucose transfer into the pleural space and impaired efflux of acidic by-products, due to an abnormal pleural membrane (tumor or fibrosis) [42]. This pathogenetic mechanism may underlie the inverse correlation between MMP-9 levels and either pH or glucose levels in malignant pleural effusions [37, 43]. However, our data do not provide evidence for a causal relationship between MMP-9 production and pleural changes. The mechanisms related to the observed correlations remain to be elucidated.

MMPs and TIMPs are implicated in invasion and metastasis of malignant tumors [15, 16]. In particular, MMP-2 and MMP-9 have been shown to be mainly involved in the invasion of carcinomas, having a high affinity for type IV collagen, which is a major component of the basement membranes. Overexpression of MMP-2 and MMP-9 has also been reported in lung cancer. Furthermore, their expression has been found to be correlated with clinicopathologic factors and prognosis [17, 18, 44, 45]. In this study, the gelatinolytic activities of MMP-2 were high in the malignant pleural effusions of lung cancer, but significant differences were not found compared with the other groups, and even transudates showed considerable activities. In this respect, the presence of MMP-2 in pleural effusions seemed constitutional, in concordance with a previous report [7]. Concentrations and gelatinolytic activities of MMP-9 in malignant pleural effusions of lung cancer appeared higher than those in transudates, although lower than those in tuberculous pleural effusions. By immunohistochemistry, most of the tumor cells in pleural tissues expressed MMP-9, although at low levels, suggesting their roles in MMP-9 production.

MMP-9 activity is down-regulated by TIMP-1, which binds to the active and latent forms of MMP-9 and is the major TIMP secreted by mononuclear phagocytes [2]. The balance between local MMP-9 and TIMP-1 concentrations critically determines the net proteolytic activity. MMP-9 and TIMP-1 have been shown to be regulated independently by different upstream signaling pathways in human monocytic cells in response to Mtb [11]. It was reported that while MMP-9 levels were increased in the cerebrospinal fluid of tuberculous meningitis compared with bacterial and viral meningitis, TIMP-1 levels were not different among the groups [46]. In our study, TIMP-1 concentrations in pleural effusions were also similar in the three groups, and its expression was relatively generalized throughout all cell types in the immunohistochemistry of pleural tissues. These findings suggest the constitutional expression of TIMP-1, unlike the inducible patterns observed for MMP-9. However, it is also noteworthy that a significant correlation was found between pleural fluid MMP-9 and TIMP-1 concentrations. This correlation may imply a causal relationship between MMP-9 and TIMP-1. This speculative explanation, however, needs to be addressed in further investigations.

We acknowledge the limitations of this study. First, cytokines in the pleural fluid originate from both systemic circulation (via vascular leakage) and cellular release in the pleural cavity. We were not able to attribute the relative contribution of each of these sources to the overall cytokine levels in our samples. Also, the biological role of MMP-9 in pleural effusions could not be addressed from our methodologies, nor could we establish a causal relationship between increased MMP-9 and the development of pleural effusions.

In conclusion, we found that MMP-9 was significantly increased in tuberculous pleural effusions compared with transudates and malignant pleural effusions of lung cancer. Among the many cell types that are involved in the production of MMP-9, the monocyte/macrophage lineage, particularly the epithelioid cells of granulomas, were found to be the major cellular source of MMP-9.

  References

1.

Nagase H, Woessner JF Jr: Matrix metalloproteinases. J Biol Chem 1999;274:21491-21494.

2.

Baker AH, Edwards DR, Murphy G: Metalloproteinase inhibitors: Biological actions and therapeutic opportunities. J Cell Sci 2002;115:3719-3727.

3.

Stetler-Stevenson WG: Dynamics of matrix turnover during pathologic remodeling of the extracellular matrix. Am J Pathol 1996;148:1345-1350.

4.

Woessner JF Jr: Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J 1991;5:2145-2154.

5.

Zucker S, Mirza H, Conner CE, Lorenz AF, Drews MH, Bahou WF, Jesty J: Vascular endothelial growth factor induces tissue factor and matrix metalloproteinase production in endothelial cells: Conversion of prothrombin to thrombin results in progelatinase A activation and cell proliferation. Int J Cancer 1998;75:780-786.

6.

Hurewitz AN, Zucker S, Mancuso P, Wu CL, Dimassimo B, Lysik RM, Moutsiakis D: Human pleural effusions are rich in matrix metalloproteinases. Chest 1992;102:1808-1814.

7.

Eickelberg O, Sommerfeld CO, Wyser C, Tamm M, Reichenberger F, Bardin PG, Soler M, Roth M, Perruchoud AP: MMP and TIMP expression pattern in pleural effusions of different origins. Am J Respir Crit Care Med 1997;156:1987-1992.

8.

Gordon S, Cohn ZA: Bacille Calmette-Guérin infection in the mouse. Regulation of macrophage plasminogen activator by T lymphocytes and specific antigen. J Exp Med 1978;147:1175-1188.

9.

Gordon S, Todd J, Cohn ZA: In vitro synthesis and secretion of lysozyme by mononuclear phagocytes. J Exp Med 1974;139:1228-1248.

10.

Quiding-Jarbrink M, Smith DA, Bancroft GJ: Production of matrix metalloproteinases in response to mycobacterial infection. Infect Immun 2001;69:5661-5670.

11.

Friedland JS, Shaw TC, Price NM, Dayer JM: Differential regulation of MMP-1/9 and TIMP-1 secretion in human monocytic cells in response to Mycobacterium tuberculosis. Matrix Biol 2002;21:103-110.

12.

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.

13.

Rivera-Marrero CA, Schuyler W, Roser S, Roman J: Induction of MMP-9 mediated gelatinolytic activity in human monocytic cells by cell wall components of Mycobacterium tuberculosis. Microb Pathog 2000;29:231-244.

14.

Hoheisel G, Sack U, Hui DS, Huse K, Chan KS, Chan KK, Hartwig K, Schuster E, Scholz GH, Schauer J: Occurrence of matrix metalloproteinases and tissue inhibitors of metalloproteinases in tuberculous pleuritis. Tuberculosis (Edinb) 2001;81:203-209.

15.

Kleiner DE, Stetler-Stevenson WG: Matrix metalloproteinases and metastasis. Cancer Chemother Pharmacol 1999;43(suppl):S42-S51.

16.

Himelstein BP, Canete-Soler R, Bernhard EJ, Dilks DW, Muschel RJ: Metalloproteinases in tumor progression: The contribution of MMP-9. Invasion Metastasis 1994;14:246-258.

17.

Thomas P, Khokha R, Shepherd FA, Feld R, Tsao MS: Differential expression of matrix metalloproteinases and their inhibitors in non-small cell lung cancer. J Pathol 2000;190:150-156.

18.

Iizasa T, Fujisawa T, Suzuki M, Motohashi S, Yasufuku K, Yasukawa T, Baba M, Shiba M: Elevated levels of circulating plasma matrix metalloproteinase 9 in non-small cell lung cancer patients. Clin Cancer Res 1999;5:149-153.

19.

Yamamura T, Nakanishi K, Hiroi S, Kumaki F, Sato H, Aida S, Kawai T: Expression of membrane-type-1-matrix metalloproteinase and metalloproteinase-2 in nonsmall cell lung carcinomas. Lung Cancer 2002;35:249-255.

20.

Marshall BC, Santana A, Xu QP, Petersen MJ, Campbell EJ, Hoidal JR, Welgus HG: Metalloproteinases and tissue inhibitor of metalloproteinases in mesothelial cells. Cellular differentiation influences expression. J Clin Invest 1993;91:1792-1799.

21.

Goetzl EJ, Banda MJ, Leppert D: Matrix metalloproteinases in immunity. J Immunol 1996;156:1-4.

22.

Johnatty RN, Taub DD, Reeder SP, Turcovski-Corrales SM, Cottam DW, Stephenson TJ, Rees RC: Cytokine and chemokine regulation of proMMP-9 and TIMP-1 production by human peripheral blood lymphocytes. J Immunol 1997;158:2327-2333.

23.

Light RW, Macgregor MI, Luchsinger PC, Ball WC Jr: Pleural effusions: The diagnostic separation of transudates and exudates. Ann Intern Med 1972;77:507-513.

24.

Alexandrakis MG, Kyriakou D, Alexandraki R, Pappa KA, Antonakis N, Bouros D: Pleural interleukin-1 beta in differentiating transudates and exudates: Comparative analysis with other biochemical parameters. Respiration 2002;69:201-206.

25.

Tahhan M, Ugurman F, Gozu A, Akkalyoncu B, Samurkasoglu B: Tumour necrosis factor-alpha in comparison to adenosine deaminase in tuberculous pleuritis. Respiration 2003;70:270-274.

26.

Aoe K, Hiraki A, Murakami T, Eda R, Maeda T, Sugi K, Takeyama H: Diagnostic significance of interferon-gamma in tuberculous pleural effusions. Chest 2003;123:740-744.

27.

Atanackovic D, Block A, de Weerth A, Faltz C, Hossfeld DK, Hegewisch-Becker S: Characterization of effusion-infiltrating T cells: Benign versus malignant effusions. Clin Cancer Res 2004;10:2600-2608.

28.

Leber TM, Balkwill FR: Zymography: A single-step staining method for quantitation of proteolytic activity on substrate gels. Anal Biochem 1997;249:24-28.

29.

Yao PM, Maitre B, Delacourt C, Buhler JM, Harf A, Lafuma C: Divergent regulation of 92-kDa gelatinase and TIMP-1 by HBECs in response to IL-1beta and TNF-alpha. Am J Physiol 1997;273:L866-L874.

30.

Ara T, Kusafuka T, Inoue M, Kuroda S, Fukuzawa M, Okada A: Determination of imbalance between MMP-2 and TIMP-2 in human neuroblastoma by reverse-transcription polymerase chain reaction and its correlation with tumor progression. J Pediatr Surg 2000;35:432-437.

31.

Hurewitz AN, Wu CL, Mancuso P, Zucker S: Tetracycline and doxycycline inhibit pleural fluid metalloproteinases. A possible mechanism for chemical pleurodesis. Chest 1993;103:1113-1117.

32.

Saunders BM, Frank AA, Orme IM: Granuloma formation is required to contain bacillus growth and delay mortality in mice chronically infected with Mycobacterium tuberculosis. Immunology 1999;98:324-328.

33.

Smith D, Hansch H, Bancroft G, Ehlers S: T-cell-independent granuloma formation in response to Mycobacterium avium: Role of tumour necrosis factor-alpha and interferon-gamma. Immunology 1997;92:413-421.

34.

Orme IM, Cooper AM: Cytokine/chemokine cascades in immunity to tuberculosis. Immunol Today 1999;20:307-312.

35.

Tsuda T, Dannenberg AM Jr, Ando M, Rojas-Espinosa O, Shima K: Enzymes in tuberculous lesions hydrolyzing protein, hyaluronic acid and chondroitin sulfate: A study of isolated macrophages and developing and healing rabbit BCG lesions with substrate film techniques - The shift of enzyme pH optima towards neutrality in 'intact' cells and tissues. J Reticuloendothel Soc 1974;16:220-231.

36.

Hrabec E, Strek M, Zieba M, Kwiatkowska S, Hrabec Z: Circulation level of matrix metalloproteinase-9 is correlated with disease severity in tuberculosis patients. Int J Tuberc Lung Dis 2002;6:713-719.

37.

Light RW: Clinical practice. Pleural effusion. N Engl J Med 2002;346:1971-1977.

38.

Saren P, Welgus HG, Kovanen PT: TNF-alpha and IL-1beta selectively induce expression of 92-kDa gelatinase by human macrophages. J Immunol 1996;157:4159-4165.

39.

Black RA, Rauch CT, Kozlosky CJ, Peschon JJ, Slack JL, Wolfson MF, Castner BJ, Stocking KL, Reddy P, Srinivasan S, Nelson N, Boiani N, Schooley KA, Gerhart M, Davis R, Fitzner JN, Johnson RS, Paxton RJ, March CJ, Cerretti DP: A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells. Nature 1997;385:729-733.

40.

Leib SL, Leppert D, Clements J, Tauber MG: Matrix metalloproteinases contribute to brain damage in experimental pneumococcal meningitis. Infect Immun 2000;68:615-620.

41.

Aleman C, Alegre J, Segura RM, Armadans L, Surinach JM, Varela E, Soriano T, Recio J, Fernandez De Sevilla T: Polymorphonuclear elastase in the early diagnosis of complicated pyogenic pleural effusions. Respiration 2003;70:462-467.

42.

Good JT Jr, Taryle DA, Sahn SA: The pathogenesis of low glucose, low pH malignant effusions. Am Rev Respir Dis 1985;131:737-741.

43.

Heffner JE, Nietert PJ, Barbieri C: Pleural fluid pH as a predictor of pleurodesis failure: Analysis of primary data. Chest 2000;117:87-95.

44.

Michael M, Babic B, Khokha R, Tsao M, Ho J, Pintilie M, Leco K, Chamberlain D, Shepherd FA: Expression and prognostic significance of metalloproteinases and their tissue inhibitors in patients with small-cell lung cancer. J Clin Oncol 1999;17:1802-1808.

45.

Urbanski SJ, Edwards DR, Maitland A, Leco KJ, Watson A, Kossakowska AE: Expression of metalloproteinases and their inhibitors in primary pulmonary carcinomas. Br J Cancer 1992;66:1188-1194.

46.

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.

  Author Contacts

Kwang Joo Park, MD, PhD
Laboratory of Cell Signaling
Building 50, NHLBI, NIH
Bethesda, MD 20892 (USA)
Tel. +1 301 496 3694, Fax +1 301 480 0357, E-Mail parkk2@mail.nih.gov

  Article Information

Received: February 2, 2004
Accepted after revision: July 21, 2004
Number of Print Pages : 10
Number of Figures : 6, Number of Tables : 0, Number of References : 46

  Publication Details

Respiration (International Journal of Thoracic Medicine)

Vol. 72, No. 2, Year 2005 (Cover Date: March-April 2005)

Journal Editor: C.T. Bolliger, Cape Town
ISSN: 0025-7931 (print), 1423-0356 (Online)

For additional information: http://www.karger.com/res

  Drug Dosage / Copyright

Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.