Departments of 1 Internal Medicine and 2 Pathology, University of Nebraska Medical Center, Omaha, Nebraska 68198-5300
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ABSTRACT |
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Fibroblasts can contract collagen gels, a process
thought to be related to tissue remodeling. Because epithelial cells
are also involved in repair responses, we postulated that human
bronchial epithelial cells (HBECs) could cause contraction of collagen
gels. To evaluate this, HBECs were plated on the top of native type I
collagen gels and were incubated for 48 h. After this, the gels were
released and floated in LHC-9-RPMI 1640 for varying times, and gel size was measured with an image analyzer. HBECs caused a marked
contraction of the gels within 24 h; the area was reduced by 88 ± 4% (P < 0.01). The degree of gel
contraction was dependent on cell density; 12,500 cells/cm2 resulted in maximal
contraction, and half-maximal contraction occurred at 7,500 cells/cm2. Contraction varied
inversely with the collagen concentration (91 ± 1% with 0.5 mg/ml
collagen vs. 43 ± 5% with 1.5 mg/ml collagen). In contrast to
fibroblasts that contract gels most efficiently when cast into the gel,
HBEC-mediated contraction was significantly (P < 0.01) more efficient when cells
were on top of the gels rather than when cast into the gels.
Anti-1-integrin antibody
blocked HBEC-mediated contraction by >50%, whereas
anti-
2-,
anti-
3-, anti-
v-,
anti-
v
5-,
anti-
2-, or
anti-
4-integrin antibody was without effect. The combination of
anti-
1-integrin antibody and an
anti-
-subfamily antibody completely blocked gel contraction induced
by HBECs. In contrast, anti-cellular fibronectin antibody did not block
HBEC-induced gel contraction, whereas it did block fibroblast-mediated
gel contraction. In summary, human airway epithelial cells can contract
type I collagen gels, a process that appears to require integrins but
may not require fibronectin. This process may contribute to airway
remodeling.
integrin; repair; remodeling
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INTRODUCTION |
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IN MOST TISSUES, injury leads to the initiation of a distinct set of repair responses. These responses include not only the recruitment and proliferation of parenchymal cells but also the production and remodeling of the extracellular matrix. In this context, fibroblasts have been reported to mediate the contraction of three-dimensional gels composed of type I collagen (9, 12). This has been suggested as a model of the contraction that frequently develops during the formation of scar or intraparenchymal fibrosis that may occur in many tissues.
Epithelial cells are known to be active participants in repair responses. This includes responding to mediators of repair by covering epithelial defects, by proliferation, and by differentiation into new epithelium. In addition, epithelial cells can produce mediators that can drive mesenchymal cell recruitment, proliferation, matrix production, and remodeling (2, 7, 11). It is not fully understood to what degree epithelial cells may directly participate in remodeling processes, although contraction of collagen gels has been reported to be caused by epithelial cells from the skin and from the retina (13, 14).
In the airways, injury of the epithelium is likely a frequent event associated with diseases such as asthma or chronic bronchitis. Consistent with the occurrence of epithelial injury, asthma is associated with a thickened deposit of interstitial collagen located directly beneath the epithelial cells and chronic bronchitis is associated with peribronchial fibrosis. Importantly, limitation of expiratory airflow has been associated not only with the presence of peribronchial fibrosis but also with a reduction in the size of airways. It would be of potential pathogenetic significance, therefore, if airway epithelial cells could participate in wound contraction and fibrosis. The current study, therefore, was designed to determine if human bronchial epithelial cells (HBECs) can contract collagen gels.
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MATERIALS AND METHODS |
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Materials
Type I collagen gels [rat tail tendon collagen (RTTC)] were made from collagen extracted from rat tail tendons by a previously published method (8). Briefly, tendons were excised from rat tails, and the tendon sheath and other connective tissues were carefully removed. After repeated washing with tris(hydroxymethyl)aminomethane (Tris)-buffered saline (0.9% NaCl and 10 mM Tris, pH 7.5) and 95% ethanol, type I collagen was extracted in 6 mM acetic acid. Protein concentration was determined by weighing a lyophilized aliquot from each lot of collagen solution. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis routinely demonstrated no detectable proteins other than type I collagen.Monoclonal anti-human 2 (clone
P1E6)-,
3 (clone P1B5)-,
v (clone VNR 147)-,
1 (clone P4C10)-,
2 (clone P4Ha)-,
4 (clone 3E1)-, and
v
5
(clone P1F6)-integrin antibodies were purchased from GIBCO BRL (Grand
Island, NY). Monoclonal anti-cellular fibronectin antibody and
anti-plasma fibronectin antibody were purchased from Sigma Chemical
(St. Louis, MO). Monoclonal anti-human cytokeratin mouse antibody (MNF
116/M821) and anti-human vimentin mouse antibody (Va/MO725) were
purchased from DAKO (Denmark). Avidin-biotin complex kit (Vectastain)
was purchased from Vector Laboratories (Burlingame, CA).
Tissue culture supplements and media were purchased from GIBCO.
HBECs
HBECs were obtained from an autopsy by a modification of a previously published method (5). Briefly, bronchial epithelial cells were cultured under serum-free conditions using LHC-9-RPMI 1640 (a 1:1 mixture of LHC-9 and RPMI 1640; see Ref. 6). Cells were plated on collagen-coated tissue culture dishes (Vitrogen 100; Collagen, Palo Alto, CA) at 37°C in a humidified atmosphere of 5% CO2. Cells were passaged one time a week at a 1:4 ratio. Cells between passages 4 and 9 were used for experiments.Collagen Gel Contraction Assay
To determine if HBECs could contract native type I collagen gels, gels were prepared by mixing RTTC, distilled water, and 4× concentrated phenol red-free Dulbecco's modified Eagle's medium (DMEM) so that the final mixture resulted in 0.75 mg/ml of collagen, a physiological ionic strength, and 1× DMEM. Then, 0.5-ml aliquots of the mixture were cast into each well of 24-well tissue culture plates (Falcon). After gelation was complete, generally within 20 min at room temperature, freshly trypsinized HBECs suspended in LHC-9-RPMI 1640 were plated on the top of the gels. The gels were then incubated for 48 h at 37°C in a 5% CO2 atmosphere. In experiments designed to determine the incubation time of HBEC culture on gels on contraction, this interval was varied. After this incubation, the gels were gently released from the 24-well tissue culture plates and were transferred to 60-mm tissue culture dishes (Falcon) that contained 5 ml of prewarmed LHC-9-RPMI 1640. This results in floating collagen gels with epithelial cells attached to the upper surface. The gels were then incubated on a rocking platform (Bellco Biotechnology, Vineland, NJ) at 37°C in a 5% CO2 atmosphere to prevent reattachment of the gels to the bottom of the culture dishes. The area of each gel was measured with an Optomax V image analyzer (Optomax, Burlington, MA) at various times. Data are expressed as the percentage of gel area compared with the original gel size, and each data point was calculated as the mean ± SE for three identically treated gels.Effect of Anti-Integrin Antibodies on the Collagen Gel Contraction by HBECs
Collagen gels were prepared and HBECs were plated on top of the gels at a density of 12,500 cells/cm2 as described above. They were then incubated at 37°C in a 5% CO2 atmosphere for 5 h to allow the cells to attach to the surface of the gels. After this, a 1:1,000 dilution of anti-Effect of Anti-Cellular Fibronectin Antibody and Anti-Plasma Fibronectin Antibody on HBECor Fibroblast-Mediated Type I Collagen Gel Contraction
Freshly trypsinized HBECs or human fetal lung (HFL1) fibroblasts were incubated with or without a 1:500 dilution of anti-cellular fibronectin antibody, anti-plasma fibronectin antibody, and anti-human IgGComparison of the Gel Contraction by Cells on Top of Gels or Inside Gels
Gels with cells plated on top were prepared by adding 12,500 cells/cm2 of HBECs or HFL1 fibroblasts to the top of polymerized collagen gels. Gels with cells embedded within the gel were prepared by suspending 3.0 × 105 cells/ml of HBECs or HFL1 fibroblasts in the collagen solution and then casting 0.5 ml/well into a 24-well plate. After 48 h, the gels were released and were suspended in either LHC-9-RPMI 1640 or serum-free DMEM on a rocking platform. After 24 h, the area of each gel was measured by an image analyzer.Immunohistochemistry
Collagen gels were prepared, and 12,500 cells/cm2 HBECs or HFL1 fibroblasts were plated on the top of the gels and were incubated at 37°C in a 5% CO2 atmosphere. After 48 h, the gels were fixed in situ by a 4% formaldehyde solution. The gels were then washed thoroughly with phosphate-buffered saline (PBS). After this, the cells were permeabilized with 0.5% Triton X-100 for 10 min and then were washed with PBS. The cells were then treated with 1.5% normal horse serum for 1 h and were stained with a 1:50 dilution of anti-cytokeratin or anti-vimentin antibodies for 3 h. The gels were washed overnight with PBS and were allowed to react with biotinylated anti-mouse IgG horse antibody (following the manufacturer's instructions) for 2 h. Gels were washed again and were allowed to react with avidin-horseradish peroxidase for 1 h. Gels were washed overnight again and were exposed to diaminobenzidine for 3-5 min. Then they were washed with distilled water for 1 h followed by counterstaining with hematoxylin for 1 min. Then the gels were placed on slides and were mounted with glass coverslips for photographs.Statistical Analysis
All numerical values are expressed as means ± SE of three separate determinations. Groups of data were evaluated by analysis of variance. Data that appeared to be statistically significant were compared by Student's t-test. Values of P < 0.05 were considered significant. ![]() |
RESULTS |
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HBECs Cause Type I Collagen Gel Contraction
To determine if HBECs could contract native collagen gels, HBECs were plated on the top of gels and were incubated for 48 h to allow the cells to attach. The gels were then released and were incubated for an additional 24 h. Under these conditions, HBECs caused a marked contraction of the collagen gels; the area was reduced by 88 ± 4% after 24 h (Fig. 1). This was clearly a property of the HBECs because collagen gels incubated in culture medium without cells retained their original size (Fig. 1).
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Effect of Cell Density on HBEC-Mediated Contraction of Type I Collagen Gel
To investigate the effect of cell density on gel contraction, various numbers of HBECs were used for gels of varying size. On gels cast in 24-well tissue culture plates, 12,500, 6,250, and 3,125 HBECs/cm2 contracted the gels 88 ± 4, 43 ± 1, and 15 ± 2%, respectively. Gels with 1,500 cells/cm2 or less did not contract the gels significantly (Fig. 2). Gels cast in 12-well plates showed similar results (data not shown).
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Time Course of HBEC-Mediated Type I Collagen Gel Contraction
To observe the time course of collagen gel contraction by HBECs, gels containing 0.75 mg/ml collagen were prepared. HBECs were plated on the top of the gels and were cultured for 48 h. The gels with the cells adherent to the top of the gels were then released and were floated in 60-mm dishes. Contraction occurred rapidly, with most of the reduction in size occurring within 6 h and being nearly complete in 24 h (Fig. 3). A similar time course of contraction was observed with both 12,500 and 6,250 cells/cm2 plated on gels, despite the fact that the smaller cell density resulted in much less overall contraction.
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Effect of Collagen Concentration on HBEC-Mediated Collagen Gel Contraction
Increasing concentrations of collagen are known to inhibit fibroblast-mediated gel contraction. To determine if collagen concentration exerts such an effect on HBEC-mediated contraction, we prepared collagen gels with different concentrations of collagen, plated HBECs on top and, after 48 h, observed their contraction. Consistent with fibroblast-mediated collagen gel contraction, the higher the concentration of type I collagen in the gel, the less the HBECs contracted the gels (Fig. 4).
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Effect of Incubation Time of HBECs During the Anchored Phase on the Subsequent Collagen Gel Contraction
Although in most experiments HBECs were allowed to attach for 24-48 h, we observed that HBECs plated on the top of collagen gels had attached within 3 h. To determine if incubation time on the surface of the gels affected contraction rate, gels were released after 3, 6, 24, and 48 h. HBECs incubated on anchored gels for 3 or 6 h contracted collagen gels but more slowly and to a lesser extent than cells incubated for 24 or 48 h (Fig. 5).
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Role of Epithelial Cell and Matrix Interaction on Collagen Gel Contraction
Integrins.
To study the possible mechanism of gel contraction by HBECs, the role
of different subfamilies of integrins was investigated. We found that
anti-1-integrin antibody alone
could block the collagen gel contraction mediated by HBECs by >50%,
whereas the anti-
2-integrin
antibody alone inhibited minimally (Fig.
6). Nevertheless, the combination of
anti-
2-integrin antibody and anti-
1-integrin antibody could
completely block the collagen gel contraction by HBECs (Fig. 6).
Additionally, neither anti-
2-, anti-
4-,
anti-
v-,
anti-
3-, nor
anti-
v
5-integrin
antibody alone blocked the gel contraction by HBECs; nevertheless, the combination of anti-
v- or
anti-
3- with
anti-
1-integrin antibody could
completely block the collagen gel contraction by HBECs (Table 1).
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Fibronectins. HFL1 fibroblast-mediated collagen gel contraction was significantly blocked by anti-cellular fibronectin antibody (Fig. 7A). Nevertheless, neither anti-cellular fibronectin antibody nor anti-plasma fibronectin antibody blocked the HBEC-mediated type I collagen gel contraction (Fig. 7B).
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Comparison of HBEC- With HFL1 Fibroblast-Mediated Gel Contraction
To compare the gel contraction mediated by epithelial cells or fibroblasts, we cultured HFL1 fibroblasts or HBECs either on the top of gels or in the gels. After 24 h, HBECs on the top of collagen gels had contracted the gels to <40% of original size (Fig. 8). HBECs in the gel caused significantly less contraction. In contrast, fibroblasts contracted the collagen gels more effectively when they were embedded in the gels than when they were cultured on the top of gels (Fig. 8).
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Immunohistochemical Staining of HBECs and HFL1 Fibroblasts Cultured on the Top of Collagen Gels
To exclude the possibility that the contraction caused by HBEC cultures was due to contaminating fibroblasts, we examined markers for mesenchymal cells (Fig. 9). After being cultured on the top of native type I collagen gels for 48 h, HBECs were attached to the upper surface of the gel. These cells stained strongly for cytokeratin but stained faintly for vimentin. In contrast, HFL1 fibroblasts were strongly stained for vimentin, whereas they were negative for cytokeratin.
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DISCUSSION |
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In both fibrosis and wound healing, fibroblast-mediated contraction of connective tissue matrix is believed to contribute to tissue reorganization. Epithelial cells also play a prominent role in tissue repair. The current study demonstrates that HBECs can cause the contraction of native type I collagen gels and thus may also contribute to tissue remodeling after injury. Epithelial cell-mediated collagen gel contraction is dependent on cell density and is inversely dependent on the collagen concentration in the gel. Cells allowed to attach and spread for 24-48 h before gels were released contracted gels more rapidly than cells in gels released 3-6 h after plating. Integrin-mediated adhesion processes appear to be required. In contrast, anti-cellular fibronectin antibody that inhibited fibroblast-mediated gel contraction had no effect on epithelial cell-mediated collagen gel contraction. The current study, therefore, demonstrates that epithelial cells can cause the contraction of native type I collagen gels. Epithelial cells may, therefore, contribute to tissue remodeling during repair processes.
HBEC contraction of native type I collagen gels resembles fibroblast-mediated contraction in a number of respects, whereas it differed in others. Both HBEC- and fibroblast-mediated contraction increase with increasing cell density and decrease with increasing collagen concentration. Similarly, both appear to depend on integrin-mediated adhesion (12). Although controversial, it has been reported that antibodies to cellular fibronectin can inhibit fibroblast-mediated contraction of native type I collagen gels (1), a result we have been able to confirm. In contrast, antibodies to cellular fibronectin had no effect on epithelial cell-mediated collagen gel contraction, suggesting these two cell types contract the gels through different mechanisms.
In addition to the differential sensitivity to anti-fibronectin antibody, two other lines of evidence suggest that our results were not due to contaminating fibroblasts in our epithelial cell preparations. First, epithelial cells were most effective in mediating contraction when plated on top of gels, whereas fibroblasts were most effective when plated within the gel. Second, staining of fibroblasts with monoclonal antibodies demonstrated strong vimentin staining and absent keratin staining, in marked contrast to epithelial cells that demonstrated strong keratin staining and weak to absent staining for vimentin. Thus the contraction observed in the present study appears to be due to epithelial cells, not contaminating fibroblasts.
Consistent with a role for epithelial cells in modulating tissue reorganization through contraction, other investigators have reported contractile activity in epithelial cells in culture. Schafer et al. (14) have reported that keratinocytes can contract collagen gels, and Sakamoto et al. (13) have reported that retinal pigment epithelial cells can also mediate collagen gel contraction. Similar to the results of the current study, Schafer et al. (14) found that keratinocytes did not require fibronectin to support collagen gel contraction. Whether the ability to contract three-dimensional extracellular matrix structures will prove to be a generalized property of epithelial cells or whether this represents a property of specialized epithelia will require additional studies with additional cell strains. Moreover, future studies will be required to determine what processes can regulate this property of epithelial cells. The current study, however, supports the potential role for epithelial cells in tissue remodeling and, importantly, suggests that this may prove to be an important feature in determining tissue architecture in the airways.
It is generally thought that fibroblast-mediated contraction of collagen gels involves attachment of fibroblasts to extracellular collagen fibers through integrin-mediated adhesion complexes and the generation of a tensile force through active participation of cytoskeletal elements (4, 12). Our results are consistent with a similar process operating for epithelial cell-mediated contraction of collagen gels. There are other possible mechanisms for epithelial cells to cause gel contraction, however.
Epithelial cells, when grown as a monolayer, can transport ions and water selectively in one direction and, therefore, could generate an osmotic gradient. Although it is possible that such an osmotic activity could contribute to epithelial cell-mediated contraction of collagen gels, several observations suggest that this is unlikely. First, the collagen gels that are undergoing contraction are floating in an isotonic tissue culture medium. It is unlikely that epithelial cells could generate much of an osmotic gradient under these conditions. Second, contraction of the collagen gel begins within 1 h of plating the epithelial cells on the surface of the collagen gels. Again, because contraction begins before a dense monolayer is formed, it seems unlikely that a strong osmotic gradient could be established. Contraction, however, begins concurrently with epithelial cell spreading, and it therefore seems reasonable to suggest that contraction is a result of the changes in cell shape. An additional possibility is that some secreted product of epithelial cells could be contributing to collagen gel contraction. Conditioned medium from epithelial cells, however, was unable to induce contraction of collagen gels (data not shown).
Under normal circumstances in vivo, epithelial cells rest on a matrix composed not of type I collagen but on a true basement membrane. Preliminary studies have suggested that basement membrane components derived from a murine sarcoma (Matrigel) can inhibit epithelial cell-mediated contraction of collagen gels (data not shown). Whether this depends on extracellular matrix components or whether it is the result of some other factor in the crude Matrigel preparation remains to be determined. The results of the current study, however, clearly demonstrate that epithelial cells can mediate the contraction of a collagen gel. In a setting in which epithelial damage takes place, it is possible that basement membrane could be destroyed and epithelial cells may be induced to migrate over a surface composed of interstitial collagens. Consistent with such a possibility, epithelial cells derived from both the lung and the skin have been noted to be more migratory over surfaces composed of interstitial collagens than over surfaces composed of basement membrane components (3, 10). The results of the current study suggest that if epithelial cells reepithelialize a surface composed of interstitial collagen, they may be able to cause the remodeling of that tissue. It is not difficult to imagine that such a process may contribute to airway narrowing in conditions such as chronic obstructive pulmonary disease. A similar process may contribute to the condensation of interstitial collagen frequently observed beneath the epithelium in chronic asthma. In the current study, epithelial cells were plated on the surface of the collagen gel, and gel contraction was measured 24-48 h later. Although the present study clearly demonstrates that cells under such conditions can mediate collagen gel contraction, the current study does not resolve the issue of whether this is a property of normal resting cells or of cells responding to an injury. That is, the culture conditions used, namely freshly plated cells that are growing and spreading to achieve confluence, may represent a model of airway injury. Whether epithelial cells will manifest differential activity with respect to contraction of their subjacent connective tissue as a function of their state of injury and/or repair is an important one. The system described in this report may prove to be valuable in assessing such questions.
The ability of an organ to repair after injury is an important determinant of whether injury results in long-term disability. Current concepts suggest that the epithelial cells that line the airways have considerable capacity to participate in repair responses. These cells can not only migrate to cover a defect and proliferate and differentiate to restore epithelial integrity but can also release mediators that can drive the accumulation, proliferation, and matrix production of subjacent fibroblasts (2, 7, 11). Through such mechanisms, airway epithelial cells may contribute to the restoration of normal tissue architecture after injury or may contribute to the development of airway fibrosis. The current study suggests another mechanism by which epithelial cells can participate in this process, namely through the direct remodeling of extracellular collagenous matrix. If this process plays a role in airway diseases such as asthma or bronchitis, it could represent a novel therapeutic target in those diseases.
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FOOTNOTES |
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Address for reprint requests: S. I. Rennard, Pulmonary and Critical Care Medicine Section, Dept. of Internal Medicine, Univ. of Nebraska Medical Center, 600 South 42nd St., Omaha, NE 68198-5300.
Received 22 July 1997; accepted in final form 17 September 1997.
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