Institut National de la Santé et de la Recherche Médicale Unité U296 and Département de Physiologie, Faculté de Médecine, 94010 Créteil; and Département de Biologie Cellulaire et Moléculaire, Commissariat à l'Energie Atomique, 91191 Gif-sur-Yvette, France
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ABSTRACT |
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In this study, we addressed the question of
whether human bronchial epithelial cells (HBECs) contribute to the
regulation of 92-kDa gelatinase activity by secreting tissue inhibitor
of metalloproteinase (TIMP)-1. We investigated expression of 92-kDa gelatinase and TIMP-1 in response to lipopolysaccharide (LPS) and to
the proinflammatory cytokines interleukin (IL)-1 and tumor necrosis
factor (TNF)-
. Confluent HBECs from explants were cultured in
plastic dishes coated with type I and III collagen. We demonstrated that TIMP-1 was expressed at both the protein and mRNA levels by
primary cultures of HBECs. Gelatin zymography of HBEC-conditioned media
showed that exposure of HBECs to LPS, IL-1
, or TNF-
induced a
twofold increase in the latent form of 92-kDa gelatinase production, as
well as its activation. Also, quantitative reverse transcriptase (RT)-polymerase chain reaction (PCR) demonstrated a twofold increase in
the 92-kDa mRNA level in response to both cytokines. In contrast, TIMP-1 production evaluated by immunoblotting was unchanged in the
presence of LPS and IL-1
and was clearly decreased in the presence
of TNF-
. Quantitative RT-PCR demonstrated that TIMP-1 mRNA levels
remained unchanged in response to LPS or IL-1
but decreased by 70%
in the presence of TNF-
. All of these results strongly suggest that
the control mechanisms regulating the expression of 92-kDa gelatinase
and TIMP-1 by HBECs in response to inflammatory stimuli are divergent
and result in an imbalance between 92-kDa gelatinase and TIMP-1 in
favor of the metalloproteinase. Such an imbalance may contribute
significantly to acute airway inflammation.
matrix metalloproteinases; inflammatory cytokines; lung; tissue
inhibitor of metalloproteinase-1; human bronchial epithelial cells; interleukin-1; tumor necrosis factor-
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INTRODUCTION |
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MATRIX METALLOPROTEINASES (MMPs) play an important role in the proteolytic degradation of the extracellular matrix (ECM), both in physiological processes, such as tissue remodeling, angiogenesis, wound healing, etc., and during pathological events, such as tumor invasion and metastatic progression (18). Regulation of the role of MMPs in ECM degradation occurs mainly at three levels. At the transcriptional level, MMP expression is precisely controlled by various cytokines acting through positive or negative regulatory elements of its genes. After secretion from the cell, MMP activity is controlled both by proteolytic activation of latent proenzymes and by interactions with their specific inhibitors.
Naturally occurring inhibitors, tissue inhibitors of metalloproteinases (TIMPs), are important controlling factors in the actions of MMPs and tissue destruction in disease processes. The balance between the levels of activated MMPs and of free TIMPs plays an important role in the regulation of MMP activity, effectively controlling the amount of connective tissue breakdown. Alterations in this equilibrium affect angiogenesis (20), cell growth (13), cell differentiation (9), and embryonic development (21). Four distinct TIMP molecules (TIMP-1, TIMP-2, TIMP-3, and TIMP-4) have been isolated, cloned, and characterized in several species (2, 6, 12, 17). The major inhibitor, a glycoprotein with an apparent molecular size of 30 kDa called TIMP-1, is encoded by a single 0.9-kb mRNA. It is produced by a variety of human tissues and by many tumor cell lines (5, 19, 32). TIMP-1 inhibits MMPs by forming a 1:1 complex (stoichiometry) with activated interstitial collagenase (MMP-1), stromelysin (MMP-3), 72-kDa gelatinase (MMP-2), 92-kDa gelatinase (MMP-9), and pro-MMP-9 in a noncovalent fashion.
Expression of the genes for MMPs and TIMPs in different normal and
tumor cells is regulated by a variety of physiological and
pharmacological agents in a manner that may be cell-type and tissue
specific. The effects of interleukin (IL)-1, tumor necrosis factor
(TNF)-
, and lipopolysaccharide (LPS) on the biosynthesis and
secretion of MMPs and TIMPs have been studied in a variety of cell
lines. These factors induce the production of high levels of some MMPs.
The activity of MMPs is also controlled by TIMPs, which also depend on
the presence of the same cytokines in the microenvironment. It has been
shown that synthesis and secretion of TIMPs occur in several types of
MMP-producing cells, such as human alveolar macrophages, monocytes,
skin fibroblasts, and some tumoral cells (4, 19, 28, 32). In a previous
study (34), we demonstrated that primary cultures of human bronchial
epithelial cells (HBECs) constitutively express major 92-kDa matrix
gelatinase and minor 72-kDa gelatinase, both of which may be involved
in basement membrane degradation and remodeling. We therefore addressed the question of whether HBECs also contribute to the regulation of the
extracellular activity of these enzymes via secretion of TIMP-1.
In this study, we investigated the basal expression of TIMPs by primary
cultures of HBECs, as well as the effects on 92-kDa gelatinase and
TIMP-1 expression of Escherichia coli
LPS endotoxin and of the proinflammatory cytokines IL-1 and TNF-
.
We found that HBECs secreted not only 92-kDa gelatinase but also TIMP-1 under basal conditions. In the presence of IL-1
, TNF-
, or LPS, 92-kDa gelatinase activity increased sharply, whereas TIMP-1 was unchanged or diminished in response to LPS and to both cytokines. The
imbalance between 92-kDa gelatinase and local TIMP-1 activities from
HBECs in inflammatory pathological conditions suggests that metalloproteinases from HBECs may play a prominent role in the pathological remodeling of the bronchial epithelium in response to
inflammatory events.
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MATERIALS AND METHODS |
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Cell Cultures
Human bronchial epithelial biopsies were obtained by fibroscopy in 18 patients investigated for bronchopulmonary carcinoma. Biopsies were taken at a distance from the tumor. For each specimen, pathological examination confirmed that the bronchial mucosa was normal. All procedures were reviewed and approved by the Hospital Henri Mondor Committee for the Protection of Rights of Human Subjects, and written informed consent was obtained to authorize the study.HBECs were cultured as previously described (34). Briefly, two or three explants (~0.5 × 0.5 mm in size) were placed on sterile plastic dishes coated with collagen G matrix (type I and III collagen; PolyLabo). The explants were covered with 600 µl of Dulbecco's modified Eagle's medium (DMEM)-F-12 (1:1) medium (Life Technologies) and were incubated for 24 h. Two milliliters of culture medium were then added to each dish. Culture medium consisted of serum-free DMEM-F-12 (1:1) supplemented with 2% Ultroser G (Sepracor), 1% antibiotics (10,000 U/ml penicillin G sodium, 10,000 µg/ml streptomycin sulfate, and 25 µg/ml amphotericin B), and 2 mM glutamine (Life Technologies). Explants were placed in a humidified incubator at 37°C under 5% CO2 in air. The culture medium was changed every 3-4 days. Explants were cultured for 2 wk until confluence of HBECs. Also, the same explants were transferred successively to new, sterile, coated plastic dishes, at 5- to 8-day intervals, to initiate new primary HBEC cultures. As described in our previous publication (34), the confluent HBECs exhibited a flat polygonal shape and were closely opposed, as is typical of cultured epithelial cells. The beating of cilia was easily identified under a light microscope as localized movement of medium over the cells. Moreover, the epithelial nature of all cultured bronchial cells was previously confirmed by staining with antibody to cytokeratin, the characteristic component of epithelial cell intermediate filaments. Also, HBEC cultures did not stain with anti-fibroblast or KP1 antibodies, indicating that contamination by nonepithelial cell types did not occur.
Normal human mammary fibroblasts cultured at confluence on plastic dishes were used as reference cells for the human TIMP-1 assay by reverse transcriptase (RT)-polymerase chain reaction (PCR). Human alveolar macrophages were recovered from bronchoalveolar lavage specimens from four patients with adult respiratory distress syndrome (ARDS) and were used as reference cells for the human 92-kDa gelatinase assay by RT-PCR.
For measurement of the gelatinases and TIMP-1 activities as well as
isolation of total cellular RNA
(RNAT), HBEC cultures were
incubated at confluence with Ultroser G-free culture medium in the
presence of 0.2% lactalbumin for 24 h. These cultures were or were not
subsequently exposed to LPS (1 µg/ml), IL-1 (100 U/ml), or TNF-
(100 U/ml; Sigma) for 24 additional hours. To establish biological
relevance, a few cell cultures were carried out for investigating the
time course response (at 6, 12, and 24 h) or dose response (10, 50, 100, and 500 U/ml) in the case of TNF-
, which has been shown to be
more reactive.
Zymography and Reverse Zymography
The HBEC culture medium was harvested and stored atActivities in the gel slabs were quantified using semiautomated image analysis (National Institutes of Health Image 1.52), which quantifies both the surface and the intensity of lysis bands after scanning the gels. Results were expressed as arbitrary units per 24 hours per 103 cells. To check that this method for measuring enzymatic activity on zymograms was linear over the range of activities in unknown samples, we evaluated activities for increasing volumes of culture medium and found that arbitrary units obtained with the image analysis system increased linearly with the volume of the samples (r = 1.00; see Ref. 10).
TIMP-1 secreted into the culture medium was detected using reverse zymography (11). Briefly, 25 µl of 20-fold concentrated conditioned media were resolved by 11.5% SDS-PAGE in the presence of 1 mg/ml porcine skin gelatin. The standard zymographic method was modified after removal of SDS from the gel by incubation of the gel for 45 min at 37°C in conditioned medium from p-aminophenylmercuric acetate-activated rabbit skin fibroblasts, which provided a source of activated metalloproteinases capable of degrading the gelatin in the gel. The gel was then incubated and stained in the same way as standard zymogram. Protection of the gelatin in the gel by the presence of TIMPs led to the production of relatively dark bands against a lighter background. Recombinant TIMP-1 was used as the reference (Valbiotech).
Immunoblotting
Aliquots of 20-fold concentrated HBEC-conditioned media were separated by 11.5% SDS-PAGE in the presence of 10%RNA Extraction
RNAT was extracted from HBECs, human macrophages, and fibroblasts using Trizol reagent (Life Technologies) according to an improvement to the single-step RNA isolation method developed by Chomczynski and Sacchi (7a). RNAT was quantified at 260/280 nm, and the integrity of the samples was checked by 1.5% agarose gel electrophoresis. Reproducible amounts of 8-15 µg RNAT were obtained from 106 cells, and aliquots were stored in sterile microfuge tubes atQuantitative RT-PCR of TIMP-1 mRNA
Primer design and synthesis. For RT-PCR experiments, sense and antisense primers were designed using previously published cDNA sequences for human TIMP-1 (6). Specific sense and antisense primers with closely similar fusion temperature values >55°C were selected as follows: sense primer 5'-GGGGACACCAGAAGTCAACCAGA-3', antisense primer 5'-CTTTTCAGAGCCTTGGAGGAGCT-3', which begin at base +140 and +517. The positions of the 5'-ends of the primers are numbered from the adenine, thymine, guanine initiation codon of the TIMP-1 gene. The TIMP-1 primer corresponded to a cDNA fragment of 400 bases. These primers (synthesized and purified by Eurogentec) were also checked for minimal self-priming and upper/lower dimer formation.Sense and antisense primers for human 92-kDa gelatinase were selected as previously described in our recent work (34).
RT step. To minimize sample handling and contamination, RT and PCR steps were performed sequentially in the same reaction tube. To a final volume of 25 µl, the following compounds were added: 3 µl of 10× PCR buffer (200 mM Tris · HCl, pH 8.3, 500 mM KCl, 15 mM MgCl2, and 1 mg/ml gelatin), 10 µl dilution buffer for RNA [10 µl of 1 M Tris, pH 8.3, 20 µl of 0.1 M dithiothreitol (DTT), 1 µl RNasin, 100 µl bovine serum albumin, and 80 µl H2O], 10 µl RNAT obtained from cultured HBECs (5, 10, and 20 ng), and 2 µl corresponding downstream primer (10 pmol). After heating for 2 min at 80°C in the thermocycler to break up secondary structures, the tubes were equilibrated at 42°C. Each sample was supplemented with 25 µl of RT mix containing 2.5 µl of 10× PCR buffer, 1.25 mM each of dATP, dCTP, dTTP, and dGTP (16 µl), 100 mM MgCl2 (1.5 µl), and 100 mM DTT (4 µl) with or without 200 units of Moloney murine leukemia virus (Life Technologies). The final volume was 50 µl. The RT reaction lasted 45 min and was carried out at 42°C to prevent excessive mispriming and possible RNA refolding. After completion of RT, the temperature was raised to 96°C for 30 s to inactivate the enzyme and to denature the RNA-DNA hybrid. The temperature was then equilibrated at 80°C.
PCR. The amplification reaction was
initiated by adding 50 µl of a mix containing 5 µl of 10× PCR
buffer, 2 µl of upper primer (10 pmol), 0.3 µl of
Taq polymerase (1.5 units; Life
Technologies), 0.3 µl of
[-32P]dCTP (3 µCi/nmol; Amersham), and 42.4 µl of
H2O. The final volume was 100 µl. Samples were overlaid with mineral oil and were subjected to the
following sequential steps: denaturation at 96°C for 30 s,
annealing at 60°C for 30 s, and extension at 72°C for 45 s. Thirty cycles were performed for TIMP-1 assay, and the last
amplification was followed by a final 10-min elongation step at
72°C.
To ensure that the amplification products were generated from the RNAT and were not contaminating cellular DNA, we performed PCR directly on RNAT that had not been subjected to the RT step. Other negative controls included PCR amplification of all the RT reagents except RNAT. A positive control for TIMP-1 mRNA expression was also included in the assay and consisted of RNAT harvested from human fibroblasts (2.5 ng). The positive control for 92-kDa gelatinase mRNA expression was RNAT harvested from human alveolar macrophages (10 ng). PCR products (3 µl) were resolved by 5% PAGE with 0.5× TBE (100 mM Tris, 90 mM boric acid, and 1 mM EDTA) and were analyzed by autoradiography. Band sizes were previously verified by 2% agarose gel electrophoresis with 0.5× TBE in the presence of molecular mass markers.
Characterization of RT-PCR products. We characterized RT-PCR products issued from HBECs and fibroblasts, after digestion with specific restriction enzymes, i.e., Nci I and Nco I (Life Technologies). The expected fragments (133 and 267 bp after digestion by Nci I, 122 and 278 bp by Nco I) were analyzed by 2% agarose gel electrophoresis.
Generation of the internal PCR standard. The quantitative RT-PCR assay that we developed required availability of a specific internal DNA standard corresponding to the TIMP-1 RNAT target. This internal DNA standard was obtained by amplification of a foreign DNA fragment from the ampicillin resistance gene in the pBluescript II SK plasmid (Stratagene), using two composite primers. Each composite primer was composed of the corresponding target gene primer sequence attached to a short segment of nucleotides that hybridized to the opposite strand of the foreign DNA fragment as follows
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To perform quantitative PCR, serial dilutions of known quantities of internal DNA standard (103-105 molecules for which a linear dose response was obtained) were added to PCR amplification tubes containing constant amounts of target RNAT. After resolution by 2% agarose gel to verify the size of the bands, PCR products were resolved by 5% PAGE. The quantities of amplified internal standard or amplified target RNAT in each tube were compared by autoradiography and were evaluated using the same semiautomated image analysis procedure that was used for zymograms. Finally, the amount of target mRNA was evaluated by interpolation between the limits of the linear standard curve.
Quantitative RT-PCR for 92-kDa gelatinase was carried out using
RNAT as previously described (34).
Briefly, 10 ng RNAT obtained either from HBECs with or without LPS, IL-1, or TNF-
exposure or
from macrophages were subjected to RT-PCR in the presence of 5 × 103 molecules of 92-kDa gelatinase
internal standard.
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RESULTS |
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Constitutive and LPS or Cytokine-Modulated Production of TIMP-1 by HBECs
Reverse zymography. Under basal conditions, reverse zymography of 20-fold concentrated media from HBECs demonstrated constitutive secretion of an ~30-kDa molecular mass band related to TIMP-1 (Fig. 1), with no lower molecular mass band (TIMP-2). TIMP-1 activity appeared to be unchanged in response to LPS and IL-1
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Western blot analysis. Both
recombinant unglycosylated human TIMP-1 (recombinant TIMP-1) used as a
positive control and 20-fold concentrated HBEC-conditioned media were
recognized by polyclonal rabbit antibody against human TIMP-1 (Fig.
2). This result confirmed that cultured
HBECs at confluence can constitutively secrete a 30-kDa protein
corresponding to TIMP-1, which was detected above by reverse
zymography. The immunoreactive band corresponded to the level of total
TIMP-1 protein because Western data were obtained in the presence of
-mercaptoethanol, which disrupts any pro-92-kDa gelatinase/TIMP-1 complexes. TIMP-1 content was unchanged
in response to LPS and IL-1
, whereas it was clearly diminished in
the presence of TNF-
. No response was observed with the same
membrane using preimmune serum (negative control). Moreover, all assays
using polyclonal anti-TIMP-3 failed to demonstrate any presence of
TIMP-3 (24-25 kDa), even in the 20-fold concentrated culture
medium and whatever the experimental conditions (basal conditions or in
the presence of LPS, IL-1
, or TNF-
.)
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Evidence of TIMP-1 mRNA expression by HBECs. RT-PCR for TIMP-1 from HBECs was successful and provided a single band of the expected size (400 bp). Also, our results clearly showed that RT-PCR was RNAT dose dependent (Fig. 3A). PCR performed directly on RNAT not subjected to the RT step and run in parallel with the test samples was negative. RT-PCR controls consisting of RNAT harvested from human mammary fibroblasts (reference cells for TIMP-1) were positive.
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Characterization of RT-PCR products from HBECs by a set of restrictive enzymes yielded two bands of the expected sizes (133 and 267 bp with Nci I and 122 and 278 bp with Nco I), thus definitively identifying the amplification products as TIMP-1 (Fig. 3B).
Development of a semiquantitative RT-PCR. We determined the optimal experimental conditions for quantifying TIMP-1 mRNA levels. For this, incremental amounts (103-107 molecules) of specific internal DNA standard (585 bp for TIMP-1) were amplified together with a constant amount (2.5 ng) of the target RNAT (Fig. 4). Two serial bands of the expected size were observed. Under our experimental conditions, and with no more than 105 molecules of internal DNA standard, the amount of amplified target remained constant, whereas the amount of amplified internal DNA standard increased linearly as a function of the initial concentration of internal DNA. For all used internal DNA standard amounts between 103 and 105 molecules, the calculated amounts of amplified target were constant. With >105 molecules of internal DNA standard, the reaction became competitive and difficult to quantify. We consequently chose to evaluate the amount of amplified target by direct interpolation to the coamplified internal DNA standard within the limits of the linear standard curve. Finally, to evaluate the TIMP-1 mRNA level, quantitative RT-PCR carried out on HBEC RNAT was performed via coamplification with 5 × 104 molecules of the corresponding specific internal DNA standard.
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Evaluation of TIMP-1 mRNA levels in HBECs using
quantitative RT-PCR: Modulation by LPS and inflammatory
IL-1 and TNF-
cytokines. Coamplification with specific internal DNA
standard and scanning analysis of autoradiograms (Fig.
5) clearly showed that HBECs produced about
five times less TIMP-1 mRNA than human fibroblasts (4.8 × 104 mRNA molecules/10 ng
RNAT for HBECs vs. 26 × 104 mRNA molecules/10 ng
RNAT for human fibroblasts). LPS-
or IL-1
-exposed HBECs exhibited similar TIMP-1 mRNA levels than
nonexposed cells (4.5 and 4.7 × 104, respectively, vs. 4.8 × 104 mRNA molecules/10
ng RNAT). In contrast, the level
of TIMP-1 mRNA decreased in response to TNF-
, with a 70% decrease
in the TIMP-1 mRNA steady-state level compared with the constitutive level (1.4 × 104 vs. 4.8 × 104 mRNA molecules/10 ng
RNAT).
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LPS- and Cytokine-Stimulated Production of 92-kDa Gelatinase by HBECs
Zymography. As expected under basal conditions (Fig. 6), the gelatinase activities investigated were detected in four forms as follows: a major band of 92 kDa produced by the proform of gelatinase B (MMP-9), a barely visible 88-kDa band corresponding to the active form of gelatinase B, and two minor bands at 72 and 68 kDa corresponding to the pro and active forms of gelatinase A (MMP-2), respectively. Production of 92-kDa gelatinase production as a latent form increased sharply (2-fold) and similarly in the presence of LPS, IL-1
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A time course study (Fig.
7A)
showed that the activity of 92-kDa gelatinase was increased in a linear
manner as a function of time (6, 12, and 24 h) after the addition of
TNF- (100 U/ml) in the culture medium. A parallel experiment
demonstrated that the 92-kDa gelatinase activity was systematically
increased about twofold in response to 10, 50, 100, or 500 U/ml
whatever the time course (Fig. 7B).
|
Evaluation of 92-kDa mRNA levels in HBECs using
quantitative RT-PCR: Modulation by LPS and inflammatory
IL-1 and TNF-
cytokines. As in our earlier study (37),
coamplification with specific internal DNA standard and scanning
autoradiogram analysis (Fig. 8) showed that
1) the level of 92-kDa mRNA from
HBECs was comparable to the level of 92-kDa gelatinase from human
alveolar macrophages (3.2 × 104 mRNA molecules/10 ng
RNAT from HBECs vs. 3.7 × 104 mRNA molecules/10 ng
RNAT from human alveolar
macrophages) and 2) the level of
92-kDa mRNA from LPS-stimulated HBECs was identical to the level from
nonstimulated HBECs (3.2 × 104 mRNA molecules/10 ng
RNAT). In contrast, compared
with nonstimulated cells, the levels of 92-kDa mRNA produced by
IL-1
- and TNF-
-stimulated HBECs were increased about twofold (5.6 × 104 vs. 3.2 × 104 mRNA molecules/10 ng
RNAT).
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DISCUSSION |
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Evidence for Presence in HBECs of TIMP-1
We previously reported that 92-kDa gelatinase expression by HBECs increased markedly in response to LPS endotoxin (34). This finding raised the question of whether 92-kDa gelatinase expression was subjected to a control mechanism involving inhibition by TIMPs produced by the same cells. Using a combination of immunological, enzymatic, and RT-PCR data, the present study provides evidence that HBECs constitutively express TIMP-1, one of the specific metalloproteinase inhibitors. TIMP-1 is found in body fluids and culture media as a secretory component of cells and has been identified in a variety of tissues and species (7). However, this is the first report of production of TIMP by HBEC. Production of both gelatinase B and TIMP-1 by the same cell type is in accordance with previous work, suggesting that progelatinase B and TIMP-1 are secreted as a complex (33). Also, production of both gelatinase B and TIMP-1 suggests very tight regulation of ECM degradation by bronchial epithelial cells.In contrast, it is of interest to note that, besides the absence of a
detectable level of TIMP-4 transcripts in the lung (12), TIMP-2 as well
as TIMP-3 was undetectable by reverse zymography and by Western blot,
respectively, even using 20-fold concentrated conditioned medium. These
results strongly suggest that TIMP-1 would be preferentially
synthesized and secreted by HBECs. Because complex formation with
TIMP-2 may occur more specifically with the 72-kDa gelatinase pro form,
the absence of TIMP-2 may be relevant to the very low level of
constitutive expression of 72-kDa gelatinase, as well as to the
almost nonexistent positive regulation of 72-kDa gelatinase in response
to LPS and to the proinflammatory cytokines IL-1 and TNF-
.
Modulation of TIMP-1 Expression by LPS, IL-1, and
TNF-
in HBECs
In contrast, quantitative RT-PCR showed a 70% decrease in the TIMP-1
mRNA level from TNF--exposed HBECs. This result is in accordance
with the diminution of TIMP-1 protein level in response to TNF-
, as
observed by immunoblotting. In contrast to immunoblotting, reverse
zymography only showed a slight decrease in TIMP-1 protein activity;
this may reflect some difficulty in estimating the exact value of data
from reverse zymography. Other authors found, on the contrary, that the
expression of TIMP-1 was markedly upregulated by TNF-
in human
myeloblastic HL-60 leukemia cells (16). These divergent results suggest
cell-type specific control mechanisms for the expression of TIMP-1.
Upregulation of 92-kDa Gelatinase for HBECs by IL-1
and TNF-
Our results clearly show that IL-1 and TNF-
enhance 92-kDa
gelatinase expression at both the protein and the mRNA levels. Also,
they confirm our recent works (34) that demonstrated marked upregulation of 92-kDa gelatinase production and activation in response
to LPS, with only minimal changes in mRNA levels due to an increase in
the half-life of the specific mRNA. Moreover, time course and
dose-response studies carried out in the presence of TNF-
clearly
demonstrate that 1) the 92-kDa
gelatinase is linearly neosynthesized by HBECs as a function of time
and is actively secreted as early as during the first 6 h of cell
culture and 2) HBECs have the
capacity to amplify the production of 92-kDa gelatinase in response to
a concentration of proinflammatory cytokine TNF-
as weak as
10
12 M, thus supporting
some physiological relevance. Indeed, the inducible upregulation of
92-kDa gelatinase from HBECs in response to the inflammatory cytokines
IL-1
and TNF-
as well as to E. coli LPS strongly supports the hypothesis that 92-kDa
gelatinase may be involved not only in the repair of damaged human
respiratory epithelium (3) but also in inflammatory pulmonary processes such as acute lung injury. Increased production of 92-kDa gelatinase by
HBECs may contribute to focal degradation of the subepithelial basement
membrane, as well as to cell-matrix disruption and to detachment of
epithelial cells.
Imbalance Between 92-kDa Gelatinase and TIMP-1 Expression by HBECs
Exposure to LPS endotoxin or to the proinflammatory cytokines IL-1In addition to elevated 92-kDa gelatinase expression by HBECs in
response to LPS, IL-1, or TNF-
, our study demonstrated that a
proportion of 92-kDa gelatinase underwent extracellular activation, as
demonstrated by increased production of the 88-kDa active form.
Gelatinase and other MMPs are secreted as latent proforms. The
conversion of latent metalloproteinases to active enzymes is an
important step for the regulation of metalloproteinase proteolytic
activity. Our results suggest that inflammatory cytokines and LPS can
induce a high rate of conversion of latent gelatinase to its active
form (30%) and that these factors are not present under normal
physiological conditions. Activation mechanisms of 92-kDa gelatinase
are somewhat controversial and may involve other MMPs such as
matrilysin (14) or free oxygen radicals (23).
In conclusion, the marked upregulation and activation of 92-kDa
gelatinase, the unchanged expression or restrictive modulation of
TIMP-1, and the absence of detectable TIMP-2 and TIMP-3 production by
HBECs in response to inflammatory conditions may induce an imbalance
between gelatinase and TIMPs in favor of the metalloproteinase, thus
promoting extensive degradation of specific macromolecular components
of the ECM underlying HBECs as well as detachment of these cells. Thus
HBECs, which are known to release a number of cytokines, such as
IL-1 and IL-8 (8), would also be capable of directly modulating the
turnover of basement membrane and ECM, particularly during inflammatory
processes such as acute lung injury.
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ACKNOWLEDGEMENTS |
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We thank Dr. François Lebargy and Dr. Bruno Housset for providing the biopsies and Sabine Hérigault and Jeanique L'Hour-Menard for technical assistance.
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FOOTNOTES |
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Address for reprint requests: C. Lafuma, Institut National de la Santé et de la Recherche Médicale Unité 296, Faculté de Médecine, 8, rue du Gl Sarrail, 94010 Créteil, France.
Received 26 December 1996; accepted in final form 2 July 1997.
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