Evaluation of basement membrane degradation during TNF-alpha -induced increase in epithelial permeability

Jean-Claude Lacherade, Andry Van De Louw, Emmanuelle Planus, Estelle Escudier, Marie-Pia D'Ortho, Chantal Lafuma, Alain Harf, and Christophe Delclaux

Institut National de la Santé et de la Recherche Médicale Unité 492 and Service de Physiologie, Explorations Fonctionnelles (Assistance Publique-Hôpitaux de Paris), Hôpital Henri Mondor, 94010 Créteil, France


    ABSTRACT
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We evaluated whether tumor necrosis factor (TNF)-alpha induces an increase in permeability of an alveolar epithelial monolayer via gelatinase secretion and basement membrane degradation. Gelatinase secretion and epithelial permeability to radiolabeled albumin under unstimulated and TNF-alpha -stimulated conditions of an A549 human epithelial cell line were evaluated in vitro. TNF-alpha induced both upregulation of a 92-kDa gelatinolytic activity (pro form in cell supernatant and activated form in extracellular matrix) and an increase in the epithelial permeability coefficient compared with the unstimulated condition (control: 1.34 ± 0.04 × 10-6 cm/s; 1 µg/ml TNF-alpha : 1.47 ± 0.05 × 10-6 cm/s, P < 0.05). The permeability increase in the TNF-alpha -stimulated condition involved both paracellular permeability, with gap formation visualized by actin cytoskeleton staining, and basement membrane permeability, with an increase in the basement membrane permeability coefficient (determined after cell removal; control: 2.58 ± 0.07 × 10-6 cm/s; 1 µg/ml TNF-alpha : 2.82 ± 0.02.10-6 × cm/s, P < 0.05). Because addition of gelatinase inhibitors [tissue inhibitor of metalloproteinase (TIMP)-1 or BB-3103] to cell supernatants failed to inhibit the permeability increase, the gelatinase-inhibitor balance in the cellular microenvironment was further evaluated by cell culture on a radiolabeled collagen matrix. In the unstimulated condition, spontaneous collagenolytic activity inhibited by addition to the matrix of 1 µg/ml TIMP-1 or 10-6 M BB-3103 was found. TNF-alpha failed to increase this collagenolytic activity because it was associated with dose-dependent upregulation of TIMP-1 secretion by alveolar epithelial cells. In conclusion, induction by TNF-alpha of upregulation of both the 92-kDa gelatinase and its inhibitor TIMP-1 results in maintenance of the gelatinase-inhibitor balance, indicating that basement membrane degradation does not mediate the TNF-alpha -induced increase in alveolar epithelial monolayer permeability.

gelatinase; tissue inhibitor of metalloproteinase-1; epithelial permeability to albumin; A549 cell line; tumor necrosis factor-alpha


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INTRODUCTION
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ACUTE RESPIRATORY DISTRESS syndrome (ARDS) is characterized by an increase in the permeability of the alveolar capillary wall, which loses its size selectivity, allowing protein-rich edema to develop.

It is now recognized that proinflammatory cytokines, particularly tumor necrosis factor (TNF)-alpha , can per se increase the permeability of the alveolar capillary barrier. Meduri and colleagues (16) found a statistical correlation between albumin and TNF-alpha concentrations in bronchoalveolar lavage fluid from ARDS patients (16). Horvath and coworkers (9) reported that TNF-alpha increased pulmonary vascular permeability independently of neutrophils in vivo. Furthermore, TNF-alpha has been shown to increase the permeability of endothelial monolayers in vitro (3).

Among factors capable of increasing the permeability of a barrier, a proteinase, 96-kDa gelatinase B, can degrade almost all basement membrane components and seems to contribute to the TNF-alpha -induced increase in endothelial permeability (21). This gelatinase belongs to the family of matrix metalloproteinases (MMPs), which is known to participate in development, malignant tumor cell invasion, wound healing, and inflammatory processes. On the basis of substrate specificity and sequence similarities, MMPs are divided into subclasses, including interstitial collagenases, stromelysins, metalloelastase, type IV collagenases or gelatinases, and membrane-type MMP. MMPs are inhibited by the four members of the tissue inhibitor of MMP (TIMP) family (TIMP-1, -2, -3, and -4). TIMP-1 and TIMP-2 are present in a soluble form, but TIMP-3 is insoluble, bound to the extracellular matrix (ECM; see Ref. 7). TIMP-1 is responsive to a variety of external stimuli such as phorbol esters, growth factors, and cytokines. TIMP-2 is for the most part constitutive. With regard to interactions between epithelia and their basement membrane, gelatinases are of particular interest since they degrade the main components of basement membranes. Two gelatinases have been described, a 72-kDa gelatinase (gelatinase A) and a 92- to 96-kDa gelatinase (gelatinase B); we previously demonstrated that both gelatinases are synthesized by alveolar epithelial cells (5). These gelatinases are secreted as inactive pro forms (72- and 92- to 96-kDa for gelatinases A and B, respectively) and are activated in the extracellular environment (68- and 88-kDa activated forms). The 92-kDa gelatinase is responsive to a variety of external stimuli such as phorbol esters, growth factors, and cytokines. The 72-kDa gelatinase is for the most part constitutive. Both activated gelatinases can be inhibited by TIMPs; moreover, TIMP-1 can also bind to the pro form of the 92-kDa gelatinase. This binary 92-kDa progelatinase-TIMP-1 complex can inhibit active MMP. We have reported a statistical correlation between the concentrations of albumin and of total activated gelatinases in the epithelial lining fluid of ARDS patients (4). Because the epithelial barrier is the major determinant of alveolar capillary wall permeability to proteins (27) and because alveolar epithelial cells can produce both gelatinases, we evaluated the role of these gelatinases in the TNF-alpha -induced increase in permeability of an alveolar epithelial barrier in vitro. Importantly, the effects of TNF-alpha stimulation were evaluated after epithelial cells had secreted their own basement membrane or when these cells were cultured on type IV collagen because we have previously shown that type IV collagen used as a matrix substratum is associated with a homeostatic phenotype and limits the ability of human bronchial epithelial cells to degrade the matrix under TNF-alpha stimulation (31). We specially assessed whether the cell supernatant constitutes an image of microcellular environmental processes to evaluate whether epithelial lining fluid similarly transduces interstitial events.


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Reagents

DMEM, glutamine, FCS, Hanks' balanced salt solution (HBSS), and antibiotics were obtained from GIBCO BRL (Cergy-Pontoise, France). LabTek chamber slides were from Nalge Nunc International (Naperville, IL), and Transwell chambers were from Costar (Badhoevedorp, The Netherlands). 125I-labeled albumin was purchased from CIS Bio International (Gif-sur-Yvette, France). Other reagents were obtained from Sigma Chemical (L'Ile d'Abeau Chêne, France).

Culture of Alveolar Epithelial Cells (A549 Cells)

A549 human alveolar epithelial cells (American Type Culture Collection, Manassas, VA) were grown to confluence in T-75 flasks in DMEM containing 10% FCS, 2 mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 2.5 mg/ml amphotericin B.

The passage number of the cell line used for our experiments was between 83 and 88. Cells were kept in liquid nitrogen at -180°C at passage 82.

Epithelial Monolayer Permeability

We used Transwell Clear inserts with a microporous filter. The filter, which was not coated, had a surface area of 1 cm2, a pore size of 0.4 µm, and a pore density of 108/cm2.

A549 cells were seeded on the inserts in a concentration of 60,000 cells/insert and allowed to grow to confluence in a 5% CO2-95% air atmosphere at 37°C. The culture medium was DMEM containing 10% FCS. Confluence was obtained 4 days later, and monolayer permeability was studied on the day after confluence was noted.

For the determination of monolayer permeability, the insert containing the epithelial monolayer served as the luminal chamber, and the well in which the insert was suspended served as the abluminal chamber.

Four hours before the permeability measurement was begun, the culture medium was changed to serum-free medium.

At the beginning of the study, 300 µl of medium without and with stimuli [tumor necrosis factor (TNF) or phorbol 12-myristate 13-acetate (PMA)] were added to the luminal chamber, and 1,500 µl of serum-free DMEM containing 125I-albumin or FITC-dextran (molecular mass 150 kDa) as a tracer were added to the abluminal chamber. The 125I-albumin and FITC-dextran concentrations were 0.5 µCi/ml and 1 mg/ml, respectively. The volumes used, 300 µl in the luminal chamber and 1,500 µl in the abluminal chamber, produced the same level of liquid in the two chambers so that there was no hydrostatic pressure difference that could have influenced the passage of albumin.

Preliminary Experiments

Epithelial permeability was evaluated based on the passage of radiolabeled albumin across the epithelial monolayer in a basal-apical direction. The A549 monolayers were incubated at 37°C in a 5% CO2-95% air atmosphere.

FITC-dextran permeability of the unstimulated epithelial monolayer was also evaluated to check that we were able to reproduce the findings of Kobayashi et al. (10), who characterized the A549 monolayer permeability to 14 peptides or proteins and 6 dextrans.

Furthermore, transepithelial resistance was also assessed in these preliminary experiments using the Epithelial Voltohmmeter EVOM (World Precision Instruments, Sarasota, FL).

Epithelial Permeability to Albumin in Unstimulated and Stimulated Conditions

In further experiments, epithelial permeability to albumin was evaluated at a single time point; the 48-h duration was selected based on preliminary studies of the time course of permeability changes. This late time point was chosen since we wanted to evaluate the permeability changes induced by ECM degradation.

To evaluate epithelial permeability, one sample from each chamber was taken after the 48-h incubation.

The 125I activity in these samples was measured in a gamma counter. A permeability coefficient was computed as previously described (10).

The permeability coefficient (PC), derived from Fick's law, was defined as
ln[<IT>1&cjs0823;  1−</IT>(V<SUB>p</SUB><IT>+</IT>V<SUB>i</SUB>)<IT>×</IT>C<SUB>i</SUB>(<IT>t</IT>)<IT>&cjs0823;  </IT>TP]<IT>=PC</IT>[<IT>A×t</IT>(V<SUB>p</SUB><IT>+</IT>V<SUB>i</SUB>)<IT>&cjs0823;  </IT>V<SUB>p</SUB><IT>×</IT>V<SUB>i</SUB>]
where Vp and Vi are the volumes in the abluminal and luminal chambers, respectively; Ci(t) is the 125I-albumin concentration in the luminal chamber at the time point t; Ni(t) is the amount of 125I-albumin in the luminal chamber at the time point t; TP is the amount of 125I-albumin in the system (luminal + abluminal chamber); A is the area of the membrane; and t is the time in seconds.

In our experimental setup, the equation above became
PC=ln<IT>{1&cjs0823;  1−</IT>[<IT>6×</IT>N<SUB>i</SUB>(<IT>t</IT>)<IT>&cjs0823;  </IT>TP]<IT>}&cjs0823;  </IT>(<IT>t×4</IT>)
Serum-free DMEM was used for the control condition. Stimulation was achieved by adding TNF-alpha to serum-free DMEM in two concentrations (100 ng/ml and 1 µg/ml) selected based on the dose-response curve for 92-kDa gelatinase induction or 10-6 M PMA. To investigate the role of gelatinase(s) in the permeability change induced by our stimulation conditions, we added an MMP inhibitor (100 ng/ml TIMP-1 or 10-6 M BB-3103, a synthetic inhibitor kindly provided by British Biotechnology, Cambridge, UK) to the chambers.

In preliminary experiments, we checked that radioactivity remained bound to the albumin after 48 h of incubation in our unstimulated and stimulated conditions.

Indirect Demonstration of the Presence of an ECM Synthesized by the A549 Cells

When the A549 cells reached confluence in the inserts, they were removed by three brief washes with HBSS followed by a 5-min incubation with 0.025 M NH4OH, as previously described (21). In preliminary experiments, the efficacy of cell removal was checked by examination under confocal microscopy, demonstrating the absence of focal contacts, and by electron microscopy, demonstrating the absence of residual basolateral plasma membrane (see above).

The passage of radiolabeled albumin across the system in the absence of cells was then measured by placing 300 µl of serum-free DMEM in the luminal chamber and 1,500 µl of serum-free DMEM containing 125I-albumin (0.25 µCi/ml) in the abluminal chamber. Samples were taken 20 h later from both chambers.

We compared this permeability coefficient with those obtained with the insert alone and with the insert containing a 5-day-old confluent epithelial cell layer.

ECM Permeability

Immediately after the collection of samples for the study of epithelial monolayer permeability under unstimulated and stimulated conditions, the inserts and wells were washed three times with HBSS to remove the experimental stimuli and radiolabeled albumin. The inserts were then placed in new wells.

The cells were removed using a 5-min incubation with 0.025 M NH4OH, and the permeability of the remaining ECM was studied as described above.

Epithelial Cell Viability

Preliminary experiments demonstrated that neither TNF-alpha nor PMA, in the concentrations used in our study, induced epithelial cell toxicity detectable by trypan blue exclusion, as previously described (2, 25).

Gelatinase-TIMP Assays

Gelatinase assay on radiolabeled substrate measures activity resulting from noncomplexed (free) activated gelatinase(s). Thus activity found in a biological sample reflects the amount of excess active gelatinase(s) compared with the amount of antiproteinase(s). In contrast, zymography, because of the effect of SDS, allows detection and quantification of both latent and activated gelatinase(s) present in free or complexed form in the biological sample.

Gelatinase assay on radiolabeled [3H]gelatin. Gelatin was radiolabeled with [3H]acetic anhydride as previously described (30). Cell-free supernatants (100 µl) prepared as described above were incubated for 48 h at 37°C with 25 µg of [3H]gelatin [400,000 counts · min-1 (cpm) · 50 µg-1] and 1 mM of 4-(2-aminoethyl)benzenesulfonyl fluoride (serine proteinase inhibitor). Gelatin degradation was determined based on the release of TCA-soluble (15% wt/vol) radioactivity.

Gelatin zymography. Cell-free supernatants were subjected to electrophoresis on 8% (wt/vol) polyacrylamide gels containing 1 mg/ml gelatin in the presence of SDS-polyacrylamide gels under nonreducing conditions. After electrophoresis, the gels were washed in 2.5% Triton X-100 for 1 h, rinsed briefly, and incubated at 37°C for 24 h in a buffer containing 100 mM Tris · HCl, pH 7.4, and 10 mM CaCl2. After incubation, the gels were stained with Coomassie blue R250 and destained in a solution of 7.5% acetic acid and 5% methanol. Negative staining indicated zones of enzymatic activity; areas of proteolysis appeared as clear bands against a blue background.

Preparation of matrix samples for gelatin zymography. To determine gelatinase content in the ECM, the cells were removed from the matrix using a wash with PBS followed by a 5-min incubation with 0.025 M NH4OH. The remaining material was solubilized by overnight incubation at 4°C in 0.5 M acetic acid. The solubilized material was precipitated by addition of NaCl to 1 M and was collected by centrifugation. The pellets were resuspended in electrophoresis sample buffer and electrophoresed as described above (21).

Detection of gelatinolytic activity in A549 cells cultured on type IV collagen-containing matrix. We slightly modified the assay developed by Lohi and Keski-Oja (14). Type IV collagen was radiolabeled with [3H]acetic anhydride as previously described (30). Radiolabeled type IV collagen (800,000 cpm/50 µg) was mixed with agarose diluted in HBSS (0.16 and 0.21% final concentrations), and 200 µl of this mixture were deposited on 0.5-ml LabTek chamber slides and allowed to solidify at 4°C. On this coating, A549 human alveolar epithelial cells were grown to confluence in 300 µl of DMEM containing 10% FCS, 2 mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 2.5 mg/ml amphotericin B. Cells were incubated at 37°C in a 5% CO2-95% air atmosphere for 48 h, at which time the cells had reached confluence. The culture medium was then aspirated, the chambers were rinsed two times with serum-free DMEM, 300 µl of FCS-free DMEM were deposited in the chambers, and the cells were incubated for an additional 48 h. Next, 200 µl of the supernatant were collected, and collagen degradation was evaluated based on the release of TCA-soluble (15% wt/vol) radioactivity. Chambers with a radiolabeled matrix but no A549 cells served as controls. To determine whether gelatinases were involved in collagen degradation, an inhibitory profile was obtained by adding to the matrix 10-6 M BB-3103 (MMP inhibitor) or 1 µg/ml TIMP-1 (natural MMP inhibitor).

ELISA for TIMP-1. Immunoreactive TIMP-1 was measured in A549 cell supernatants using a commercially available TIMP-1 ELISA kit (Amersham, Orsay, France). This ELISA kit consistently detected TIMP-1 concentrations >1 ng/ml in a linear fashion. Results are expressed as the means of duplicate assays.

Ultrastructure Studies

Transmission electron microscopy. To characterize precisely the different components involved in epithelial permeability, namely tight junctions and secreted ECM, we performed electron microscopy studies. Confluent A549 monolayers were fixed in 2.5% glutaraldehyde in 0.045 M cacodylate buffer at pH 7.4 for 2 h at 4°C. Monolayers were then postfixed in buffered 1% osmium tetroxide for 90 min, stained in 2% uranyl acetate, and dehydrated in graded ethanol solutions. Cell culture filters were removed from the inserts and embedded in Epon. Thin sections were examined with the Philips EM 301 (Endhoven, The Netherlands) electron microscope at a final magnification ranging from ×13,000 to ×36,000.

Actin filament staining. The actin cytoskeleton was visualized by rhodamine-phalloidin staining of the cell monolayers grown on filters, as described elsewhere (24). The stained preparations were examined using laser confocal microscopy with an LSM 410 inverted microscope (Zeiss, Rueil-Malmaison, France). Comparisons were made with monolayers fixed and stained with May-Grünwald Giemsa.

Statistical Analysis

Data are expressed as means ± SE. Groups of data were tested using ANOVA. Differences that appeared significant were evaluated using Student's t-test for comparing the means of multiple groups and were considered significant if P values were <0.05.


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Components of Epithelial Monolayer Permeability on Electron Micrographs

Electron microscopy examination showed that cell junctions, mainly small tight junctions at the apical membrane, loosely associated the A549 cells. A thin ECM was also uniformly evidenced beneath the cell monolayer (Fig. 1). Both of these elements, tight junctions and ECM, participate in the low transepithelial resistance (resistance of the monolayer - resistance of the microporous filter alone: 21 ± 4 Omega  · cm2, n = 5) measured in preliminary experiments.


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Fig. 1.   Electron micrograph of A549 monolayer. Cells were grown on filters. After fixation with glutaraldehyde, cells were processed as described in METHODS. A: apical side. A549 cells grew in monolayers and exhibited microvilli (MV) on their apical membrane and lamellar bodies (LB), and tight junctions (TJ) between adjacent cells were evidenced. Original magnification was ×13,000. B: apical side. Small tight junctions between adjacent cells are evidenced at higher magnification (original magnification was ×18,000). C: basal side. A549 cells grew in monolayers that adhered to the secreted extracellular matrix (ECM) through focal contacts (FC) located in basal plasma membrane. Original magnification was ×18,000.

TNF-alpha -Induced Epithelial Cell Expression of 92-kDa Gelatinolytic Activity

Gelatin substrate zymography of serum-free medium conditioned by A549 cells showed that the predominant gelatinolytic activity, expressed constitutively, had a relative molecular mass of 72 kDa. The form with a molecular mass of 92 kDa was barely detectable in the unstimulated condition. Treatment of A549 cells with TNF-alpha (0.01-1,000 ng/ml) resulted in a dose-dependant release into the medium of a 92-kDa gelatinolytic activity that plateaued at 100 ng/ml (Fig. 2). Treatment with 10-6 M PMA similarly induced 92-kDa gelatinolytic activity (both 92-kDa pro form and activated 88-kDa form). The levels of the constitutively released 72-kDa activity were not affected by exposure of A549 cells to either TNF-alpha or PMA.


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Fig. 2.   Gelatin zymography of supernatants and ECM extracts of A549 human epithelial cells. Top: cell-free supernatants were subjected to electrophoresis on 8% (wt/vol) polyacrylamide gels containing 1 mg/ml gelatin in the presence of SDS (SDS-PAGE) under nonreducing conditions. Purified human 92-kDa gelatinase B was deposited on lanes 1 and 2 (4 and 8 ng/ml, respectively). Lanes 3-9 show a dose response to tumor necrosis factor (TNF)-alpha (0.01-1,000 ng/ml), and lane 10 shows the response to 10-6 M phorbol 12-myristate 13-acetate (PMA). Marked upregulation of a 92-kDa gelatinolytic activity occurred with both stimulants and was dose dependent with TNF-alpha . By contrast, the 72-kDa gelatinolytic activity was not modified by stimulation. Both activities were abolished by incubation of the gel with 10 mM EDTA (data not shown). Bottom: ECM-bound gelatinolytic activities were examined by zymography. Extracts of matrix from unstimulated A549 cells contained a faint 72-kDa gelatinolytic activity. TNF-alpha treatment induced a dose-dependent appearance of both 92-kDa and 88-kDa forms (lanes 2 and 3).

ECM-bound gelatinolytic activities were examined by zymography. Extracts of matrix from unstimulated A549 cells contained a faint 72-kDa gelatinolytic activity. TNF-alpha treatment induced a dose-dependent appearance of both 92-kDa and 88-kDa forms.

TNF-alpha and PMA Increased Epithelial Monolayer Permeability

In permeability experiments, we used the TNF-alpha concentrations previously shown to have maximal effects on 92-kDa gelatinase secretion, namely 100 ng/ml and 1 µg/ml. TNF-alpha and PMA stimulation of epithelial cell monolayers induced a significant increase in the permeability coefficient compared with the unstimulated condition (Fig. 3). It is worth noting that PMA stimulation induced both a larger permeability increase and a larger increase in 92-kDa gelatinolytic activity in supernatants than TNF-alpha stimulation.


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Fig. 3.   Permeability coefficient of the epithelial monolayer (cell monolayer + ECM) and of residual ECM under unstimulated and stimulated conditions. Transwell clear inserts with a microporous filter were used in the permeability experiments. For the determination of monolayer permeability, the insert containing the epithelial monolayer served as the luminal chamber, and the well in which the insert was suspended served as the abluminal chamber. Epithelial permeability was evaluated based on the passage of radiolabeled albumin across the epithelial monolayer in a basal-apical direction. The A549 monolayers were incubated for 48 h under unstimulated and stimulated conditions at 37°C in a 5% CO2-95% air atmosphere. One sample from each chamber was taken after this period. The permeability coefficient of the epithelial monolayer (cell monolayer + ECM: filled bars) was then computed (see METHODS). The permeability coefficient for albumin was ~1.35 and ~0.40 × 10-6 cm/s for dextran (molecular mass 150 kDa). After collection of samples for studies of epithelial monolayer permeability under our various experimental conditions, the inserts were washed and placed in new wells. The cells were then removed, and permeability of the remaining ECM was studied (ECM: open bars). TNF-alpha induced a significant increase in the permeability coefficients of both the epithelial monolayer and the residual ECM. In contrast, PMA increased the epithelial monolayer permeability coefficient without significantly affecting the ECM permeability coefficient. In our system, residual ECM contributed ~50% of epithelial monolayer permeability (cell monolayer + ECM). *P < 0.05 and $P < 0.01 compared with the control unstimulated condition. The number of experiments is given in each bar.

Involvement of ECM in the A549 Monolayer Permeability Model

The contribution of the ECM to the barrier function of our cell monolayers was assessed. A549 cells were removed, leaving the filter coated with newly synthesized ECM. The rate of 125I-albumin flux across the ECM-coated filter was compared with that across the total monolayer system and across the uncoated filter. Results showed that the ECM produced by A549 cells contributed ~50% of the total monolayer barrier function (Fig. 3).

Effect of TNF-alpha and PMA on ECM Permeability

The contribution of ECM to monolayer barrier function was assessed under stimulated conditions (TNF-alpha and PMA) compared with the unstimulated condition. The ECM permeability coefficient was higher after TNF-alpha stimulation than in the unstimulated condition, suggesting that TNF-alpha may have induced degradation and/or reorganization of the basement membrane matrix. By contrast, in the PMA-stimulated condition, ECM permeability was not significantly different from that in the unstimulated condition (Fig. 3).

Effect of TNF-alpha and PMA on Free Gelatinolytic Activity

Free gelatinolytic activity in supernatant of the stimulated epithelial monolayer. Neither TNF-alpha nor PMA induced the release of free gelatinolytic activity into the supernatant of A549 cells. When supernatants of TNF-alpha - and PMA-stimulated A549 cells were processed with radiolabeled [3H]gelatin, no free gelatinolytic activity was evidenced (data not shown).

Free type IV collagenolytic activity in the cellular microenvironment of the epithelial monolayer. A549 cells cultured on radiolabeled matrix expressed a constitutive collagenolytic activity. When cells were cultured on a matrix containing type IV collagen, collagenolysis was evidenced. Collagenolytic activity was significantly reduced by the addition of MMP inhibitors to the collagen matrix (Fig. 4). Interestingly, collagenolytic activity did not decrease when MMP inhibitors were added to the supernatants (data not shown). Neither TNF-alpha nor PMA stimulation of A549 cells cultured on a collagen-coated matrix induced a significant increase in [3H]collagen matrix degradation compared with the unstimulated condition (Fig. 4). Lysis of the residual matrix followed by analysis of residual radioactivity showed that [3H]collagen matrix was still present at the end of all experiments in all conditions (data not shown).


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Fig. 4.   Detection of gelatinolytic activity in the cellular microenvironment of A549 cells. A mixture of 0.16% type IV collagen and 0.21% agarose diluted in Hanks' balanced salt solution (HBSS) was deposited on LabTek chamber slides and allowed to solidify at 4°C. On this coating, A549 human alveolar epithelial cells were grown to confluence. After confluence was achieved, FCS-free DMEM was deposited in the chambers, and the cells were incubated for an additional 48 h. Next, collagen degradation was determined based on the release of TCA-soluble (15% wt/vol) radioactivity in cell supernatants. Chambers with radiolabeled matrix but no A549 cells served as controls. To determine whether gelatinase(s) was involved in collagen degradation, an inhibitory profile was obtained by addition to the matrix of 10-6 M BB-3103 [matrix metalloproteinase (MMP) inhibitor] or 1 µg/ml tissue inhibitor of metalloproteinase (TIMP)-1 (natural MMP inhibitor). Spontaneous lysis of type IV collagen was evidenced [unstimulated condition, monolayer = 100% (100% = 56 ng of radiolabeled type IV collagen degraded · 10-5 cells · day-1)] and was significantly decreased by the addition of MMP inhibitor to the radiolabeled matrix. Neither TNF-alpha nor PMA significantly increased the basal collagenolytic activity. $P < 0.01 compared with the unstimulated monolayer condition. The number of experiments is given in each bar.

Gelatinase Inhibitors

Gelatinase inhibitors failed to inhibit the permeability increases induced by TNF-alpha and PMA. Addition of gelatinase inhibitors (TIMP-1 or BB-3103) to the luminal and/or abluminal chambers failed to inhibit the permeability increases induced by TNF-alpha and PMA (data not shown). Unexpectedly, the synthetic inhibitor BB-3103 induced a significant increase in permeability, even in the unstimulated condition.

TNF-alpha and PMA increased TIMP-1 secretion by A549 cells. Because TNF-alpha and PMA increased the 92-kDa gelatinolytic activity evidenced by supernatant zymography without inducing any increase in matrix degradation, we looked for an effect of TNF-alpha and PMA on the gelatinase inhibitor TIMP-1. TNF-alpha and PMA stimulation induced a dose-dependent increase in TIMP-1 production by A549 cells (Fig. 5).


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Fig. 5.   ELISA for TIMP-1. Immunoreactive TIMP-1 was measured in A549 cell supernatants using a commercially available TIMP-1 ELISA kit. Results are expressed as the means of 5 experiments. A dose-dependent increase in immunoreactive TIMP-1 was evidenced in cell-free supernatants of alveolar epithelial cells stimulated by TNF-alpha (solid line) or PMA (dashed line). *P < 0.05 compared with the unstimulated condition. $P < 0.01 compared with the unstimulated condition.

Effect of TNF-alpha or PMA on epithelial morphology. To further analyze the effect of both PMA and TNF-alpha on epithelial monolayer permeability, we used confocal microscopy to examine the effect of these stimulants on epithelial morphology. A549 cells stimulated by PMA showed a circular arrangement of actin cytoskeleton inducing intercellular gaps. TNF-alpha stimulation caused dramatic changes in A549 cell morphology, including elongation and formation of intercellular gaps (Fig. 6).


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Fig. 6.   Staining of the F-actin cytoskeleton and observation by confocal microscopy. A549 cells were incubated for 48 h in serum-free DMEM (A and D), serum-free DMEM containing 10-6 M PMA (B and E), and serum-free DMEM containing 100 ng/ml TNF-alpha (C and F). After 48 h, cells were fixed and stained with May-Grünwald Giemsa (A-C) or fixed in methanol, and 1.5 µM rhodamine-phalloidin was added (D-F). Cells were then observed by light or confocal microscopy. All monolayers (transversal middle-height section of cells) show that actin is localized as a prominent perijunctional ring (D-F). Both stimuli induced intercellular gap formation. Original magnification ×500.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Alveolar capillary wall permeability to proteins is mainly dependent on the epithelial side of the wall, where the intercellular junctions are tighter (27). The epithelial barrier is composed of alveolar epithelial cells (type I and II pneumocytes) lying on a basement membrane produced by these cells. Alveolar epithelial cells are capable not only of producing basement membrane components but also of producing and secreting proteases that degrade these components, thus allowing basement membrane turnover. The ability of alveolar cells to produce gelatinases and their inhibitors, TIMPs, has recently been demonstrated by several groups including ours (5, 20). The gelatinases are secreted as inactive pro forms (72 kDa and 92 kDa for gelatinases A and B, respectively) and are activated in the extracellular environment (68 and 88 kDa). These proteases can degrade most basement membrane components, including type IV collagen, suggesting that they are probably involved in physiological processes and perhaps also in pathophysiological processes characterized by excessive matrix degradation.

In ARDS, degradation products of type IV collagen, which is present only in basement membrane, are found in the alveolar space, indicating increased proteinase activity (11). We have found gelatinases in the alveolar spaces of ARDS patients and demonstrated a correlation between the concentrations of activated gelatinases and albumin in the epithelial lining fluid (4). These results invited an investigation of a potential role for gelatinases in the genesis of alveolocapillary wall injury. The objective of the present in vitro study was to evaluate the potential causal relationship between gelatinase production by alveolar epithelial cells and modulation of epithelial barrier permeability to albumin.

Epithelial barrier permeability to proteins is due 1) mainly to paracellular permeability dependent on the tightness of intercellular junctions and 2) to a lesser extent to basement membrane permeability. Because we wanted to explore the role of the basement membrane and its possible alteration by gelatinases, we studied an alveolar epithelial barrier composed of A549 cells, which are type II-like cells. These cells and other tumor cells are known to have leaky junctions (transepithelial resistance is only ~20 Omega  · cm2, as for endothelial monolayers; see Ref. 29) but synthesize surfactant and basement membrane components and secrete gelatinases and their inhibitors, TIMPs. Thus permeability of A549 cell monolayers is heavily dependent on basement membrane permeability. Indeed, the basement membrane contributed ~50% of total monolayer permeability in our experiments, a proportion similar to that reported by Partridge and colleagues (21) for an endothelial monolayer.

The goal of our first set of experiments was to demonstrate that TNF-alpha stimulation induced both 92-kDa gelatinase upregulation and an increase in the permeability of an epithelial monolayer.

In preliminary experiments, we assessed A549 monolayer permeability to both albumin and dextran to characterize our model and to check that our findings reproduced those of Kobayashi et al. (10), who evaluated the same A549 monolayer permeability to a wide range of proteins. The permeability coefficients of albumin and dextran (molecular mass 150 kDa) were ~1.35 and ~0.40 × 10-6 cm/s respectively, close to those previously found (~1.10 and ~0.25 × 10-6 cm/s, respectively; see Ref. 10).

The permeability of this epithelial barrier to albumin was significantly increased after stimulation by TNF-alpha in a high concentration similar to that found in the epithelial lining fluid of ARDS patients (16, 26). A similar result has been previously obtained by Li and colleagues (13) using the same monolayer. Permeability of the basement membrane itself (after cell removal) was also increased, suggesting an effect on basement membrane components. We compared the effect of TNF-alpha with that of PMA because PMA is known to induce both an increase in epithelial permeability in other cell cultures (1, 19) and upregulation of 92-kDa gelatinase synthesis by alveolar epithelial cells (15).

In addition to the increase in epithelial barrier permeability, TNF-alpha stimulation of A549 cells resulted in dose-dependent secretion of an inducible 92-kDa progelatinase, which is consistent with the expression of both 55- and 75-kDa TNF-alpha receptors on A549 epithelial cells (17). Under our conditions, 92-kDa progelatinase secretion was observed with a TNF-alpha concentration of 0.1 ng/ml and plateaued from 100 ng/ml. These high concentrations of TNF-alpha have been used by other authors to induce interleukin-6 and proteinase inhibitor secretion by the same cell line (2, 25) and are comparable to those found in the epithelial lining fluid of ARDS patients (16, 26). PMA also induced 92-kDa progelatinase secretion that was partly activated (88-kDa form). Because the activated 88-kDa form was not visualized in TNF-alpha -stimulated experiments, ECM-bound gelatinolytic activities were examined by zymography, demonstrating that TNF-alpha treatment induced a dose-dependent appearance of both 92-kDa and 88-kDa forms.

In contrast, 72-kDa progelatinase, which is constitutively expressed by alveolar epithelial cells, was not upregulated by TNF-alpha or PMA, consistent with earlier results obtained using epithelial and endothelial cells (11, 16).

Therefore, we can conclude from these experiments that 1) an upregulation of 92-kDa progelatinase secretion by A549 epithelial cells is evidenced under both PMA and TNF-alpha stimulation, 2) an activation of secreted 92-kDa progelatinase can occur in a serum-free medium, and 3) under TNF-alpha stimulation, activated 88-kDa gelatinase is probably mainly located in ECM close to its substrates [probably in focal contacts, as demonstrated for endothelial cells by Partridge et al. (22)].

In a second set of experiments, we looked for a possible causal relationship between the permeability increase and the secretion of gelatinase. In a study of the permeability of an endothelial monolayer, Partridge et al. (21) found that TNF-alpha stimulation increased both permeability and 96-kDa gelatinase secretion and that addition of exogenous endothelial 96-kDa gelatinase induced an increase in permeability that was abolished by MMP inhibitors. It is worth noting that Partridge et al. did not seek to inhibit the TNF-alpha -induced increase in permeability by adding inhibitors to the supernatants. Under our experimental conditions, addition of MMP inhibitors to the supernatants failed to abolish the permeability increase. Unexpectedly, the synthetic inhibitor BB-3103 increased epithelial permeability, perhaps as a result of the chelating properties of this hydroxamate compound (12, 29). However, before concluding that MMP inhibitors fail to inhibit the epithelial permeability increase induced by TNF-alpha , we had to check that the inhibition site was accessible to the inhibitor under our experimental conditions, i.e., that the gelatinases were close to their matrix substrates.

Consequently, we evaluated whether TNF-alpha stimulation was associated with an increase in ECM degradation. We designed experiments to evaluate the global effect of the proteinase-antiproteinase balance in the cellular microenvironment. A549 cells were cultured on a radiolabeled substrate (type IV collagen), and substrate degradation was assessed at confluence under unstimulated and stimulated conditions. Cells were cultured on type IV collagen, since we have previously shown that type IV collagen used as a matrix substratum is associated with a homeostatic phenotype and limits the ability of human bronchial epithelial cells to degrade the matrix under TNF-alpha stimulation (31). Because our aim was to evaluate the effects of TNF-alpha at the onset of injury (when alveolar capillary wall permeability increases), the underlying basement membrane at that time remains constituted by type IV collagen; in contrast, after the initial injury, when reepithelialization occurs, the ECM is constituted by type I plus III collagen, which modifies the epithelial response to TNF-alpha in terms of gelatinase/TIMP-1 expression (31).

Interestingly, our unstimulated condition was associated with type IV collagen degradation that was abolished by the addition of MMP inhibitors (TIMP-1 or BB-3103) to the coated substrate. This basal activity may reflect basement membrane renewal by alveolar epithelial cells. It was not modified by the addition of inhibitors to supernatants. These results show clearly that exogenous inhibitors added to supernatants cannot readily access their inhibition site. This basal activity could be due to 1) a faint activated 68-kDa gelatinase that cannot be detected by zymography or that remains bound to its substrate and 2) membrane-type MMPs (6). Moreover, our results emphasize that supernatant analysis does not always reflect processes occurring within the microenvironment of cells.

Unexpectedly, under our TNF-alpha - and PMA-stimulated conditions, we found no increase in substrate degradation. However, this result is in accordance with our previous results that demonstrated that type IV collagen, compared with types I plus III collagen used as a matrix substratum for human bronchial epithelial cell cultures, is associated with less upregulation of 92-kDa gelatinase under TNF-alpha stimulation, loss of activation of the pro form of 92-kDa gelatinase in cell supernatant, and maintenance of TIMP-1 production (31). Thus TNF-alpha did not induce an imbalance in favor of proteinases, a somewhat surprising finding since TNF-alpha increased 92-kDa gelatinase secretion in cell supernatants and has been shown by others to promote an excess in proteinases over proteinase inhibitors in other cell systems (30). This apparent paradox was a result of concomitant upregulation by TNF-alpha of the gelatinase inhibitor TIMP-1. Piedboeuf and colleagues (23) demonstrated that TIMP-1 mRNA upregulation occurred within 1 h after injury in rats. TNF-alpha has also been shown to upregulate elafin and secretory leukocyte proteinase inhibitor production by A549 cells (25). Furthermore, we found an excess of inhibitors compared with the proteinases elastase and gelatinase in bronchoalveolar lavage fluids of ARDS patients (4). Similarly, PMA induced TIMP-1 secretion according to its stimulatory effect on TIMP-1 mRNA induction in A549 cells (15).

Our results emphasize the importance of the mechanisms that regulate cytoskeletal contractile tension (see the effect of PMA in Fig. 6) in controlling epithelial permeability. Interestingly, the prominent perijunctional ring localization of F-actin has been previously described in other epithelia (8, 28). Our finding that the TNF-alpha -induced increase in epithelial monolayer permeability was not related to basement membrane degradation leaves open the mechanism underlying this phenomenon. The alveolar epithelial cells used in our study exhibited morphological changes quite similar to those reported for TNF-alpha -stimulated endothelial cells (21). These changes included elongation, production of stress fibers, and formation of intercellular gaps and suggest that TNF-alpha may have increased paracellular alveolar epithelial permeability, as demonstrated for other epithelial cells (18). From our experiments, it cannot be ruled out that the presence of the activated 88-kDa gelatinase could have induced a weak activity in a microcellular environment (in focal contacts; see Ref. 22) participating in the formation of the intercellular gap and thereby contributing to the increase in permeability by reducing the epithelial surface.

Similar to Partridge and colleagues (21), we found an increase in isolated basement membrane permeability in the TNF-alpha -stimulated condition. This finding may be ascribable to basement membrane reorganization without degradation. Consistent with this possibility, Partridge and colleagues found no decrease in basement membrane protein content after TNF-alpha stimulation (21). Moreover, Curtis and colleagues (3) reported that reorganization and/or disruption of the fibronectin matrix and the TNF-alpha -induced increases in endothelial permeability and proteinase expression seemed unrelated to proteolytic degradation of fibronectin within the ECM (3).

In conclusion, TNF-alpha increased alveolar epithelial monolayer permeability, consistent with its previously reported effect on endothelial monolayers. This finding strongly suggests that TNF-alpha may play a key role in making the alveolar capillary wall permeable to proteins, thus promoting alveolar edema formation independently from neutrophils. The TNF-alpha -induced epithelial permeability increase was not mediated by basement membrane degradation; both the 92-kDa gelatinase and its inhibitor, TIMP-1, were upregulated, and as a result, the gelatinase(s)-inhibitor(s) balance was unchanged.


    ACKNOWLEDGEMENTS

We thank M. C. Millepied and A. M. Vojtek (Laboratoire de Microscopie Electronique, Service d'Anatomo-pathologie, Centre Hospitalier Intercommunal de Créteil) for technical assistance in ultrastructural studies.


    FOOTNOTES

J.-C. Lacherade was supported by a Fellowship from the Fondation pour la Recherche Médicale.

Address for reprint requests and other correspondence: C. Delclaux, INSERM Unité 492, Faculté de Médecine, 8 rue du Général Sarrail, 94010 Créteil, France (E-mail: delclaux{at}im3.inserm.fr).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 8 September 2000; accepted in final form 29 January 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Lung Cell Mol Physiol 281(1):L134-L143
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