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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We evaluated whether tumor
necrosis factor (TNF)- 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-
-stimulated
conditions of an A549 human epithelial cell line were evaluated in
vitro. TNF-
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-
:
1.47 ± 0.05 × 10
6 cm/s, P < 0.05). The permeability increase in the TNF-
-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-
: 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-
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-
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-
-induced
increase in alveolar epithelial monolayer permeability.
gelatinase; tissue inhibitor of metalloproteinase-1; epithelial
permeability to albumin; A549 cell line; tumor necrosis factor-
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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)-, can per se increase the permeability of the
alveolar capillary barrier. Meduri and colleagues (16) found a statistical correlation between albumin and TNF-
concentrations in bronchoalveolar lavage fluid from ARDS patients
(16). Horvath and coworkers (9) reported that
TNF-
increased pulmonary vascular permeability independently of
neutrophils in vivo. Furthermore, TNF-
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--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-
-induced increase in permeability of an alveolar epithelial barrier in vitro. Importantly, the effects of TNF-
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-
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.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
![]() |
In our experimental setup, the equation above became
![]() |
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-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 · min1 (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
106 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. ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
|
TNF--Induced Epithelial Cell Expression of 92-kDa
Gelatinolytic Activity
|
ECM-bound gelatinolytic activities were examined by zymography.
Extracts of matrix from unstimulated A549 cells contained a faint
72-kDa gelatinolytic activity. TNF- treatment induced a
dose-dependent appearance of both 92-kDa and 88-kDa forms.
TNF- and PMA Increased Epithelial Monolayer Permeability
|
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- and PMA on ECM Permeability
Effect of TNF- and PMA on Free Gelatinolytic Activity
Free gelatinolytic activity in supernatant of the stimulated
epithelial monolayer.
Neither TNF- nor PMA induced the release of free gelatinolytic
activity into the supernatant of A549 cells. When supernatants of
TNF-
- 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- 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).
|
Gelatinase Inhibitors
Gelatinase inhibitors failed to inhibit the permeability increases
induced by TNF- 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-
and PMA (data not shown). Unexpectedly, the
synthetic inhibitor BB-3103 induced a significant increase in
permeability, even in the unstimulated condition.
TNF- and PMA increased TIMP-1 secretion by A549 cells.
Because TNF-
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-
and PMA on the
gelatinase inhibitor TIMP-1. TNF-
and PMA stimulation induced a
dose-dependent increase in TIMP-1 production by A549 cells (Fig.
5).
|
Effect of TNF- or PMA on epithelial morphology.
To further analyze the effect of both PMA and TNF-
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-
stimulation caused dramatic changes in A549 cell morphology, including elongation and
formation of intercellular gaps (Fig. 6).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
· 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- 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 × 106 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- 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-
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-
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-
receptors on A549 epithelial cells
(17). Under our conditions, 92-kDa progelatinase secretion
was observed with a TNF-
concentration of 0.1 ng/ml and plateaued
from 100 ng/ml. These high concentrations of TNF-
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-
-stimulated experiments, ECM-bound
gelatinolytic activities were examined by zymography, demonstrating
that TNF-
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- 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- stimulation, 2) an activation of secreted 92-kDa progelatinase can occur in a serum-free medium, and 3) under TNF-
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- 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-
-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-
, 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- 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-
stimulation (31). Because our aim was to evaluate the
effects of TNF-
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-
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-- 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-
stimulation,
loss of activation of the pro form of 92-kDa gelatinase in cell
supernatant, and maintenance of TIMP-1 production (31).
Thus TNF-
did not induce an imbalance in favor of proteinases, a
somewhat surprising finding since TNF-
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-
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-
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--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-
-stimulated endothelial cells (21). These
changes included elongation, production of stress fibers, and formation
of intercellular gaps and suggest that TNF-
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--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-
stimulation
(21). Moreover, Curtis and colleagues (3)
reported that reorganization and/or disruption of the fibronectin
matrix and the TNF-
-induced increases in endothelial permeability
and proteinase expression seemed unrelated to proteolytic degradation
of fibronectin within the ECM (3).
In conclusion, TNF- increased alveolar epithelial monolayer
permeability, consistent with its previously reported effect on
endothelial monolayers. This finding strongly suggests that TNF-
may
play a key role in making the alveolar capillary wall permeable to
proteins, thus promoting alveolar edema formation independently from
neutrophils. The TNF-
-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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Anderson, JM,
Balda MS,
and
Fanning AS.
The structure and regulation of tight junctions.
Curr Opin Cell Biol
5:
772-778,
1993[Medline].
2.
Crestani, B,
Cornillet P,
Dehoux M,
Rolland C,
Guenounou M,
and
Aubier M.
Alveolar type II epithelial cells produce interleukin-6 in vitro and in vivo. Regulation by alveolar macrophage secretory products.
J Clin Invest
94:
731-740,
1994[ISI][Medline].
3.
Curtis, TM,
Rotundo RF,
Vincent PA,
McKeown-Longo PJ,
and
Saba TM.
TNF--induced matrix Fn disruption and decreased endothelial integrity are independent of Fn proteolysis.
Am J Physiol Lung Cell Mol Physiol
275:
L126-L138,
1998
4.
Delclaux, C,
d'Ortho MP,
Delacourt C,
Lebargy F,
Brun-Buisson C,
Brochard L,
Lemaire F,
Lafuma C,
and
Harf A.
Gelatinases in epithelial lining fluid of patients with adult respiratory distress syndrome.
Am J Physiol Lung Cell Mol Physiol
272:
L442-L451,
1997
5.
D'Ortho, MP,
Clerici C,
Yao PM,
Delacourt C,
Delclaux C,
Franco-Montoya ML,
Harf A,
and
Lafuma C.
Alveolar epithelial cells in vitro produce gelatinases and tissue inhibitor of matrix metalloproteinase-2.
Am J Physiol Lung Cell Mol Physiol
273:
L663-L675,
1997
6.
D'Ortho, MP,
Stanton H,
Butler M,
Atkinson SJ,
Murphy G,
and
Hembry RM.
MT1-MMP on the cell surface causes focal degradation of gelatin films.
FEBS Lett
421:
159-164,
1998[ISI][Medline].
7.
Gomez, DE,
Alonso DF,
Yoshiji H,
and
Thorgeirsson UP.
Tissue inhibitors of metalloproteinases: structure, regulation and biological functions.
Eur J Cell Biol
74:
111-122,
1997[ISI][Medline].
8.
Hecht, G,
Pothoulakis C,
LaMont JT,
and
Madara JL.
Clostridium difficile toxin A perturbs cytoskeletal structure and tight junction permeability of cultured human intestinal epithelial monolayers.
J Clin Invest
82:
1516-1524,
1988[ISI][Medline].
9.
Horvath, CJ,
Ferro TJ,
Jesmok G,
and
Malik AB.
Recombinant tumor necrosis factor increases pulmonary vascular permeability independent of neutrophils.
Proc Natl Acad Sci USA
85:
9219-9223,
1988[Abstract].
10.
Kobayashi, S,
Kondo S,
and
Juni K.
Permeability of peptides in human cultured alveolar A549 cell monolayer.
Pharm Res
12:
1115-1119,
1995[ISI][Medline].
11.
Kondoh, Y,
Taniguchi H,
Taki F,
Tagaki K,
and
Satake T.
7S collagen in bronchoalveolar lavage fluid of patients with adult respiratory distress syndrome.
Chest
101:
1091-1094,
1992[Abstract].
12.
Lacaz-Viera, F.
Calcium site specificity. Early Ca2+-related tight junction events.
J Gen Physiol
110:
727-740,
1997
13.
Li, XY,
Donaldson K,
Brown D,
and
MacNee W.
The role of tumor necrosis factor in increased airespace epithelial permeability in acute lung inflammation.
Am J Respir Cell Mol Biol
13:
185-195,
1995[Abstract].
14.
Lohi, J,
and
Keski-Oja J.
Calcium ionophores decrease pericellular gelatinolytic activity via inhibition of 92-kDa gelatinase expression and decrease of 72-kDa gelatinase activation.
J Biol Chem
270:
17602-17609,
1995
15.
Mackay, AR,
Ballin M,
Pelina MD,
Nason AM,
Hartzler JL,
and
Thorgeirsson UP.
Effect of phorbol ester and cytokines on matrix metalloproteinase expression in tumor and normal cell lines.
Invasion Metastasis
12:
168-184,
1992[ISI][Medline].
16.
Meduri, GU,
Kohler G,
Headley S,
Tolley E,
Stentz F,
and
Postlethwaite A.
Inflammatory cytokines in the BAL of patients with ARDS. Persistent elevation over time predicts poor outcome.
Chest
108:
1303-1314,
1995
17.
Merolla, R,
Rebert NA,
Tsiviste PT,
Hoffmann SP,
and
Panuska JR.
Respiratory syncytial virus replication in human lung epithelial cells: inhibition by tumor necrosis factor and interferon
.
Am J Respir Crit Care Med
152:
1358-1366,
1995[Abstract].
18.
Mullin, JM,
Laughlin KV,
Marano CW,
Russo LM,
and
Soler AP.
Modulation of tumor necrosis factor-induced increase in renal (LLC-PK1) transepithelial permeability.
Am J Physiol Renal Fluid Electrolyte Physiol
263:
F915-F924,
1992
19.
Ojakian, GK.
Tumor promoter-induced changes in the permeability of epithelial cell tight junctions.
Cell
23:
95-103,
1981[ISI][Medline].
20.
Pardo, A,
Ridge K,
Uhal B,
Sznajder JI,
and
Selman M.
Lung alveolar epithelial cells synthetize interstitial collagenase and gelatinases A and B in vitro.
Int J Biochem Cell Biol
29:
901-910,
1997[ISI][Medline].
21.
Partridge, CA,
Jeffrey JJ,
and
Malik AB.
A 96-kDa gelatinase induced by TNF- contributes to increased microvascular endothelial permeability.
Am J Physiol Lung Cell Mol Physiol
265:
L438-L447,
1993
22.
Partridge, CA,
Phillips PG,
Niedbala MJ,
and
Jeffrey JJ.
Localization and activation of type IV collagenase/gelatinase at endothelial focal contacts.
Am J Physiol Lung Cell Mol Physiol
272:
L813-L822,
1997
23.
Piedboeuf, B,
Johnston CJ,
Watkins RH,
Hudak BB,
Lazo JS,
Cherian MG,
and
Horowitz S.
Increased expression of tissue inhibitor of metalloproteinases (TIMP-I) and metallothionein in murine lungs after hyperoxic exposure.
Am J Respir Cell Mol Biol
10:
123-132,
1994[Abstract].
24.
Planus, E,
Galiacy S,
Matthay MA,
Laurent V,
Gavrilovic J,
Murphy G,
Clérici C,
Isabey D,
Lafuma C,
and
d'Ortho MP.
Role of collagenase in mediating in vitro alveolar wound repair.
J Cell Sci
112:
243-252,
1999
25.
Sallenave, JM,
Shulmann J,
Crossley J,
Jordana M,
and
Gauldie J.
Regulation of secretory leukocyte proteinase inhibitor (SLPI) and elastase-specific inhibitor (ESI/Elafin) in human airway epithelial cells by cytokines and neutrophilic enzymes.
Am J Respir Cell Mol Biol
11:
733-741,
1994[Abstract].
26.
Suter, PM,
Suter S,
Girardin E,
Roux-Lombard P,
Grau GE,
and
Dayer JM.
High bronchoalveolar levels of tumor necrosis factor and its inhibitors, interleukin-1, interferon, and elastase, in patients with adult respiratory distress syndrome after trauma, shock, or sepsis.
Am Rev Respir Dis
145:
1016-1022,
1992[ISI][Medline].
27.
Wangensteen, OD,
Wittmers LE,
and
Johnson JA.
Permeability of the mammalian blood-gas barrier and its components.
Am J Physiol
216:
719-727,
1969[ISI][Medline].
28.
Welsh, MJ,
Shasby CM,
and
Husted RM.
Oxidants increase paracellular permeability in a cultured epithelial cell line.
J Clin Invest
76:
1155-1168,
1985[ISI][Medline].
29.
Winton, HL,
Wan H,
Cannell MB,
Gruenert DC,
Thompson P,
Garrod DR,
Stewart GA,
and
Robinson C.
Cell lines of pulmonary and non-pulmonary origin as tools to study the effects of house dust mite proteinases on the regulation of epithelial permeability.
Clin Exp Allergy
28:
1273-1285,
1998[ISI][Medline].
30.
Yao, PM,
Buhler JM,
d'Ortho MP,
Lebargy F,
Delclaux C,
Harf A,
and
Lafuma C.
Expression of matrix metalloproteinase gelatinases A and B by cultured epithelial cells from human bronchial explants.
J Biol Chem
271:
15580-15589,
1996
31.
Yao, PM,
Delclaux C,
d'Ortho MP,
Maitre B,
Harf A,
and
Lafuma C.
Cell-matrix interactions modulate 92-kD gelatinase expression by human bronchial epithelial cells.
Am J Respir Cell Mol Biol
18:
813-822,
1998