Human Pleural Effusions Are Rich in Matrix Metalloproteinases

doi:10.1378/chest.102.6.1808

Chest

Volume 102, Issue 6, December 1992, Pages 1808-1814

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We identified and characterized type IV collagenase and gelatinase activity in pleural fluid from 32 patients. The capacity to substantially degrade type IV collagen was demonstrated in every pleural sample. Comparable results were also noted for the degradation of a radiolabeled gelatin substrate. Gelatin gel zymography of the pleural fluids revealed two prominent zones of lysis at 66 kDa and 92 kDa. These were identified by specific polyclonal antibodies as human matrix metalloproteinases MMP-2 and MMP-9. The concentration of MMP-2 in pleural fluid, as measured by enzyme-linked immunoassay, averaged 1,622 ng/ml whereas those of MMP-9 were 210 ng/ml. Substrate degradation activity was compared in both serum and pleural fluid from three patients and found to be similar. In serum this enzymatic activity was primarily due to MMP-9 whereas in pleural fluid, the predominant gelatinase was MMP-2. This was confirmed by immunoassay that showed that MMP-2 levels were two to five times higher in pleural fluid than in serum. We conclude that substantial amounts of MMP-2 and, to a lesser degree, MMP-9 are present in pleural effusions. The bioactivity and the immunoactivity of these enzymes did not help to distinguish among pleural fluids characterized as transudates, nonmalignant exudates, or malignant exudates. The differences in the distribution of these enzymes in pleural fluid and blood suggest that their presence is not due simply to the ultrafiltration of plasma, but rather to synthesis by the resident cells at the pleural surfaces.

Section snippets

Reagents

Trypsin-N-tosyl-L-phenylalanylchloromethyl ketone (trypsin-TPCK), 5-alpha-p-tosyl-L-Iysine chloromethylketone (TLCK), soybean trypsin inhibitor (SBTI), phenylmethylsulfonyl fluoride (PMSF), and edetic acid were obtained (Sigma Chemical Co, St Louis, Mo). Hydrofluor was obtained (National Diagnostics, South Somerville, NJ). Gelatin-sepharose was purchased (Pharmacia-LKB, Piscataway, NJ). Biotinylated affinity purified goat anti-mouse IgG antibody and alkaline phosphatase conjugated streptavidin

Substrate Degradation Assays

Human pleural fluids exhibited substantial type IV collagenolytic activity. The average activity for all unmodified samples was 1.0±0.8 µg of substrate degraded per hour of incubation per milliliter of sample (mean±SEM). A comparison was made of the type IV collagenolytic activity of the pleural fluids grouped according to three clinical categories: malignant effusion, nonmalignant exudative effusion, and transudate; no significant differences in activity were observed by the method of analysis

Discussion

The gelatinases that we uncovered in pleural fluid had activity against both type IV collagen and heat-denatured type I collagen (gelatin). This activity was documented by three distinct methods. First, the capacity to degrade either type IV collagen or gelatin was measured using a soluble radiolabeled substrate assay that detects overall enzymatic activity in the sample. Second, gelatin zymography was used to enumerate the various gelatinases in pleural fluid and indicate their relative

References (26)

SM Wilhelm et al.
SV40-transformed human lung fibroblasts secrete a 92-kDA type IV collagenase which is identical to that secreted by normal human macrophages
J Biol Chem
(1989)
Y Okada et al.
A metalloproteinase from human synovial fibroblasts that digests connective tissue matrix components: purification and characterization
J Biol Chem
(1986)
D Moscatelli et al.
Tetradecanolyl phorbol acetate stimulates latent collagenase production by cultured human endothelial cells
Cell
(1980)
MS Hibbs et al.
Biochemical and immunological characterization of the secreted forms of human neutrophil gelatinase
J Biol Chem
(1985)
S Garbisa et al.
Transient expression of type IV collagenolytic metalloproteinase by human mononuclear phagocytes
J Biol Chem
(1986)
IE Collier et al.
H-ras oncogene-transformed human bronchial epithelial cells (TBE-1) secrete a single metalloprotease capable of degrading basement membrane collagen
J Biol Chem
(1988)
P Bohlen et al.
Fluorometric assay of proteins in the nanogram range
Arch Biochem Biophys
(1973)
V Lefebvre et al.
Enzymatic evaluation of procollagenase and collagenase inhibitors in crude biological media
Biochim Biophys Acta
(1989)
A Sellers et al.
Neutral metalloproteinases of rabbit bone: separation in latent forms of distinct enzymes that when activated degrade collagen, gelatin and proteolglycans
Biochem J
(1978)
B Birkedal-Hansen et al.
Monoclonal antibodies to human fibroblast procollagenase: inhibition of enzymatic activity, affinity purification of the enzyme, and evidence for clustering of epitopes in the NH₂-terminal end of the activated enzyme
Biochemistry
(1988)

GP Stricklin et al.

Human skin collagenase: isolation of precursor and active forms from both fibroblast and organ cultures

Biochemistry

(1977)

G Murphy et al.

Gelatinase (type IV collagenase) immunolocalization in cells and tissues: use of an antiserum to rabbit bone gelatinase that idenfifies high and low Mr forms

J Cell Sci

(1989)

H Tschesche et al.

Interstitial collagenase, gelatinase and a specific type IV/V (basement membrane) collagen degrading proteinase from human leukocytes

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Vascular endothelial growth factor correlates with matrix metalloproteinase-9 in the pleural effusion
2004, Respiratory Medicine
Vascular endothelial growth factor (VEGF) is a potent, multifunctional cytokine that contributes to angiogenesis and inflammation. Matrix metalloproteinase-9 (MMP-9) is one of the major proteolytic enzymes that degrade various components of the extracellular matrix. Few data are available on the potential relationship between VEGF and MMP-9 in the accumulation of pleural effusion. We examined levels of VEGF and MMP-9 by means of enzyme immunoassay, zymographic analysis, and Western blot analysis in the patients with liver cirrhosis, tuberculosis, or lung cancer. The levels of VEGF and MMP-9 were significantly increased in the pleural fluids and sera of patients with tuberculosis and were even higher in patients with lung cancer compared with the patients with liver cirrhosis. A significant correlation was established between the level of VEGF and the level of MMP-9 in the pleural effusion. These results suggest that overproduction of VEGF and MMP-9 is associated with accumulation of the pleural effusion in tuberculosis and lung cancer. The relationship between VEGF and MMP-9 in the pleural effusion may have a role in the pathogenesis of pleural fluid formation.
Ovarian carcinoma cells in serous effusions show altered MMP-2 and TIMP-2 mRNA levels
2001, European Journal of Cancer
Citation Excerpt :
However, the relative contribution of each of these cellular components to the effusion cell population may vary considerably in different specimens. Two studies of pleural effusions have previously demonstrated the presence of MMPs and TIMPs in both malignant and non-malignant exudates, as well as in transudates [22,23]. However, the methods employed (enzyme linked immunosorbent assay (ELISA), zymography and immunoblotting) did not render possible the detection of the cellular source of these proteins.
The expression of matrix metalloproteinases (MMP) and their inhibitor TIMP-2 in serous effusions from patients with ovarian carcinoma and its association with clinico-pathological parameters were analysed. The findings in carcinoma cells in effusions were compared with corresponding primary and metastatic lesions. Sixty-six effusions and 96 tissue sections were stained for MMP-1, MMP-2 and MMP-9 applying immunohistochemistry (IHC) and analysed for MMP-2, MMP-9 and TIMP-2 expression using mRNA in situ hybridisation (ISH). MMP-2 and MMP-9 mRNA levels in 30 effusions were subsequently analysed using reverse transcription- polymerase chain reaction (RT-PCR). MMP and TIMP expression was detected in both carcinoma and mesothelial cells in effusions. The levels were consistently higher in malignant cells, significantly so for MMP-1 (P=0.016) and MMP-2 (P=0.036) proteins, as well as for TIMP-2 mRNA (P=0.008). In tissue sections, MMP-1, MMP-2 and MMP-9 protein expression was mostly localised to tumour cells, while MMP-2, MMP-9 and TIMP-2 mRNA were predominantly detected in stromal cells. Adenocarcinoma cells in effusions showed a significant upregulation of MMP-2 expression compared with primary tumours, with a concomitant downregulation of TIMP-2. RT-PCR demonstrated the presence of MMP-2 and MMP-9 in 28/30 and 0/30 specimens, respectively. MMP and TIMP are thus mainly synthesised by cancer cells in effusions, while stromal cells have a similar role in solid tumours. MMP-1 and MMP-2 production predominates over that of MMP-9 in effusions. Increased MMP-2 and reduced TIMP-2 levels are seen in ovarian carcinoma cells in effusions, possibly marking the acquisition of a metastatic phenotype.
Occurrence of matrix metalloproteinases and tissue inhibitors of metalloproteinases in tuberculous pleuritis
2001, Tuberculosis
Objective: Matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) have been found in high concentrations in pleural effusions. Because MMP and TIMP may play a part in the causation of the fibrosis seen in tuberculous (TB) pleuritis their occurrence was examined.
Design: Pleural effusion fluid and plasma concentrations of MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, TIMP-1 and TIMP-2 were determined by ELISA in 21 patients with TB pleuritis. To adjust for the total protein content, respective ratios were calculated. Activities of MMP-2 and MMP-9 were measured by gelatine zymography and the MMP-9/MMP-2 ratios calculated. Pleural effusions and plasma of 15 patients with congestive heat failure (CHF) and plasma of 15 healthy persons (CON) served as controls.
Results: Immunoreactive pleural fluid concentrations of MMP-1, MMP-2, MMP-8, and MMP-9 were higher in TB compared to CHF, but plasma concentrations were not different between the groups. TB pleural fluid concentrations of MMP-1, MMP-2, TIMP-1, and TIMP-2 were higher compared to TB plasma. MMP-3 was found in trace amounts only. The MMP-9/total protein ratios in pleural fluid were higher in TB compared to CHF (0.4492±0.1633 vs 0.0364±0.0145, P<0.005) but the TIMP-1 ratios were lower (139.0±28.7 vs 517.8±183.7, P<0.0005). In TB pleural fluid vs TB plasma, the respective MMP-1, MMP-2, TIMP-1, and TIMP-2 ratios were increased (0.46±0.10 vs 0.17±0.02; 25.2±2.8 vs 4.2±0.9; 139.0±28.7 vs 27.8±8.2; 0.67±0.13 vs 0.18±0.04, P<0.0005 each). Gelatine zymography demonstrated MMP-2 and MMP-9 bands of different brightness in TB effusions but in CHF effusions the MMP-9 band was barely visible. The MMP-9/MMP-2 effusion ratios were therefore higher in TB compared to CHF (0.46±0.15 vs 0.05±0.04,P <0.0005).
Conclusion: Compartmentalized MMP-1, MMP-2, TIMP-1, and TIMP-2 and, compared to CHF, a surplus of MMP-1, MMP-2, MMP-8, and MMP-9 in the pleural space obviously contribute to the fibrotic reactions in TB pleuritis.
Grooved surface topography alters matrix-metalloproteinase production by human fibroblasts
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Development of gelatin methacrylate (GelMa) hydrogels for versatile intracavitary applications
2022, Biomaterials Science
Profile of Metalloproteinases and Their Association with Inflammatory Markers in Pleural Effusions
2016, Lung

View all citing articles on Scopus

Supported by a Merit Review Grant from the Veterans Administration.

revision accepted April 10.

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Chest

Clinical InvestigationsHuman Pleural Effusions Are Rich in Matrix Metalloproteinases

Section snippets

Reagents

Substrate Degradation Assays

Discussion

J Biol Chem

J Biol Chem

Cell

J Biol Chem

J Biol Chem

J Biol Chem

Arch Biochem Biophys

Biochim Biophys Acta

Neutral metalloproteinases of rabbit bone: separation in latent forms of distinct enzymes that when activated degrade collagen, gelatin and proteolglycans

Biochem J

Monoclonal antibodies to human fibroblast procollagenase: inhibition of enzymatic activity, affinity purification of the enzyme, and evidence for clustering of epitopes in the NH2-terminal end of the activated enzyme

Biochemistry

Human skin collagenase: isolation of precursor and active forms from both fibroblast and organ cultures

Biochemistry

Gelatinase (type IV collagenase) immunolocalization in cells and tissues: use of an antiserum to rabbit bone gelatinase that idenfifies high and low Mr forms

J Cell Sci

Interstitial collagenase, gelatinase and a specific type IV/V (basement membrane) collagen degrading proteinase from human leukocytes

Clinical Investigations
Human Pleural Effusions Are Rich in Matrix Metalloproteinases

Monoclonal antibodies to human fibroblast procollagenase: inhibition of enzymatic activity, affinity purification of the enzyme, and evidence for clustering of epitopes in the NH₂-terminal end of the activated enzyme