Keratinocyte growth factor can enhance alveolar epithelial repair by nonmitogenic mechanisms

Kamran Atabai1, Masanobu Ishigaki1, Thomas Geiser2, Iris Ueki1, Michael A. Matthay1,3, and Lorraine B. Ware4

Departments of 1 Medicine and 3 Anesthesia, Cardiovascular Research Institute, University of California, San Francisco 94143-0130; 2 Division of Pulmonary Medicine, University Hospital, CH-3010 Bern, Switzerland; and 4 Division of Pulmonary and Critical Care Medicine, University of California, Los Angeles, California 94122


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Pretreatment with keratinocyte growth factor (KGF) ameliorates experimentally induced acute lung injury in rats. Although alveolar epithelial type II cell hyperplasia probably contributes, the mechanisms underlying KGF's protective effect remain incompletely described. Therefore, we tested the hypothesis that KGF given to rats in vivo would enhance alveolar epithelial repair in vitro by nonproliferative mechanisms. After intratracheal instillation (48 h) of KGF (5 mg/kg), alveolar epithelial type II cells were isolated for in vitro alveolar epithelial repair studies. KGF-treated cells had markedly increased epithelial repair (96 ± 22%) compared with control cells (P < 0.001). KGF-treated cells had increased cell spreading and migration at the wound edge but no increase in in vitro proliferation compared with control cells. KGF-treated cells were more adherent to extracellular matrix proteins and polystyrene. Inhibition of the epidermal growth factor (EGF) receptor with tyrosine kinase inhibitors abolished the KGF effect on epithelial repair. In conclusion, in vivo administration of KGF augments the epithelial repair rate of alveolar epithelial cells by altering cell adherence, spreading, and migration and through stimulation of the EGF receptor.

wound repair; adhesion; epidermal growth factor receptor; transforming growth factor-alpha ; acute lung injury


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

KERATINOCYTE GROWTH FACTOR (KGF) is a potent epithelial mitogen produced and secreted by stromal cells (17). Epithelial cells express the KGF receptor and are the main target of this growth factor. KGF is an important mediator of epithelial-mesenchymal interactions and epithelial repair in multiple organ systems, including the skin, intestine, and bladder (7, 16, 20, 31, 40, 41).

The role of KGF in wound repair in the skin has been well established in both in vitro and in vivo models. KGF isoforms increase dramatically in keratinocytes after dermal wounds (41). KGF receptor knockout mice have deficient wound repair capacity when exposed to incisional and excisional dermal grafts (42). In the lung, KGF increases the rate of in vitro wound repair in airway epithelial monolayers (37).

Several recent studies have shown that administration of KGF to rats 2-3 days before injury markedly ameliorates experimentally induced lung injury (11, 19, 25, 34, 38, 43, 44, 46). Interestingly, KGF confers no benefit when given at the time of injury. The mechanisms underlying the protective effect of KGF remain incompletely described but are probably multiple and include the mitogenic effect on alveolar epithelial type II cells, enhanced epithelial cell tight junctions, and an increase in epithelial cell production of surfactant or other proteins (40). The main objective of this study was to evaluate the nonmitogenic effects of KGF on alveolar epithelial cells that could contribute to the protective effect of KGF treatment before experimental lung injury. Therefore, rat alveolar epithelial cells were isolated 48 h after in vivo treatment with KGF and were assessed with in vitro alveolar epithelial repair assays using equal numbers of KGF-treated and control cells.

Because we found that in vivo treatment with KGF markedly augments the in vitro rate of epithelial repair independently of its mitogenic effect, we assessed the contribution of cell adhesion, spreading, and proliferation to the increased epithelial repair capacity of KGF-treated cells. Because KGF activates the epidermal growth factor (EGF) receptor pathway by increasing transforming growth factor (TGF)-alpha secretion (6, 12) and the EGF receptor pathway can predicate alveolar epithelial repair (18, 21), we hypothesized that the increase in in vitro alveolar epithelial repair after in vivo KGF treatment would depend on the EGF receptor pathway.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The studies were approved by the University of California San Francisco Committee on Animal Research.

Reagents. Elastase was purchased from Roche Diagnostics (Indianapolis, IN). Rat IgG, DNase, methylthiazoletetrazolium (MTT), fibronectin, collagen type I, III, and IV, and fatty acid-free BSA were obtained from Sigma (St. Louis, MO). KGF was a generous gift from AMGEN (Oak Cliffs, CA). MEM, FBS, penicillin, and streptomycin were obtained from the Cell Culture Facility at the University of California San Francisco. Compound 56, tyrphostin AG-1478, and the 5-bromo-2'-deoxyuridine (BrDU) labeling kit were purchased from Calbiochem (San Diego, CA). Polystyrene tissue culture (24- and 96-well) plates were purchased from Costar. Glass slides were purchased from Nunc (Rochester, NY). Diff-Quick was purchased from Dade Behring (Duedingen, Switzerland).

Intratracheal KGF instillation. Pathogen-free male Sprague-Dawley rats (120-150 g) were anesthetized with intraperitoneal administration of ketamine (50 mg/kg) and xylazine (3 mg/kg) and supplemented with oxygen. The trachea was exposed by blunt dissection, and KGF (5 mg/kg) dissolved in 1% BSA with Evans blue was instilled intratracheally (2 ml/kg) by micropuncture. Control rats received an equal volume of 1% BSA with Evans blue. Rats were housed with free access to food and water for 48 h.

Isolation of alveolar epithelial cells. KGF was instilled intratracheally, and then, 48 h later, rats were killed with intraperitoneal pentobarbitol sodium and heparin sodium (400 U/kg body wt) and were exsanguinated. Alveolar epithelial cells were isolated as previously described (13, 18). Isolated lungs were required to have Evans blue staining in all lobes (95% of isolations). Cell purity was >90% (modified Papanicolaou stain), and cell viability was >95% (trypan blue exclusion).

Cell culture. Isolated alveolar epithelial cells were suspended in 10% FBS-MEM supplemented with penicillin-streptomycin and plated at a concentration of 1 × 106 cells/well in 24-well polystyrene plates. Medium was exchanged for fresh medium at 36 h. Confluent monolayers were formed by 48 h.

Alveolar epithelial repair assay. Confluent monolayers were washed three times with MEM, and a sterile pipette was used to make a linear wound. Cells were washed extensively with MEM to remove debris. MEM with 0.1% fatty acid-free BSA and penicillin-streptomycin with or without inhibitors was added. The rate of epithelial repair over time was measured by an image analysis system, as previously described (18, 21). Images were taken at 0 and 12 h (images were also taken at 24 h in preliminary studies). All experiments were done in triplicate. The rate of alveolar epithelial repair is expressed as square micrometer per hour of the wound closed. Supernatants were collected at the end of the 12-h assay and stored at -70°C until use.

Cell adhesion. The adhesion protocol was a modification of Aumailly and Timpl (3). Freshly isolated cells suspended in 10% FBS-MEM were plated in triplicate at 50,000 cells/well in 96-well polystyrene plates with or without protein. Wells were precoated with 1% BSA, fibronectin, and collagen I, III, or IV for 2 h at 37°C, washed with PBS, blocked for 30 min with 1% BSA, and washed again with PBS before the addition of cells. At the end of the assay, cells were washed with PBS three times and fixed with 4% paraformaldehyde. Methylene blue (1%) was added for 30 min after aspiration of the paraformaldehyde. Cells were washed three times with distilled water and lysed with a 1:1 solution of 0.01 M HCl and 95% ethanol. Absorbance at 630 nm was measured. Adherence was measured at multiple time points (4-48 h after plating cells). Results are expressed as absorbance units at 630 nm.

Cell spreading. To quantify the degree of cell spreading at the wound edge, the internuclear distance between adjacent cells along the same wound edge was quantified. As cells spread, the internuclear distance between adjacent cells increases proportionally to the increase in diameter of the cells. At the end of the epithelial repair, assay plates were washed extensively with PBS and stained with Diff-Quick. The internuclear distance of cells at the migrating edge of the wound and in the intact monolayer away from the wound edge of five randomly chosen high-power fields (~25 measurements) per well (3 wells total) was measured. Images were obtained in the same manner as in the epithelial repair assay.

Proliferation assays. Proliferation during the 48 h in culture before the epithelial repair assay was measured with two different assays. The first was a tetrazolium salt-based assay (24). After isolation, cells were suspended in 10% FBS-MEM and plated at 50,000 cells/well in 96-well polystyrene plates. After 48 h, 10 µl of MTT were added at a concentration of 5 mg/ml. After 4 h, 100 µl of acid-isopropanol (0.04 N HCl in isopropanol) were added to each well, and absorbance at 570 nm was measured on a standard plate reader. The second assay used cells grown on the 24-well polystyrene plates used in the epithelial repair assay. After a confluent monolayer was formed, cells were released with trypsin and counted by a standard hemocytometer. Cell viability was >95% (trypan blue exclusion). Experiments were done in triplicate.

BrDU staining. Cells were plated on eight-well glass slides after precoating with fibronectin (20 µg/ml). Monolayers were wounded as in the epithelial repair assay. After wounding (11 h), BrDU was added for 1 h, and staining was performed according to the manufacturer's protocol. BrDU-positive cells were counted in five randomly chosen high-power fields at the wound edge and in the intact monolayer away from the wound edge.

Supernatant studies. Supernatants from control and KGF-treated cells were collected at the end of the epithelial repair assays and stored at -70°C. These supernatants were subsequently used as medium for epithelial repair assays done with new primary isolates of untreated alveolar epithelial cells.

Inhibitor studies. Confluent monolayers were washed with MEM, and a linear wound was made as in the epithelial repair assay. EGF receptor inhibitors, tyrphostin AG-1478 (1-20 µM) and compound 56 (1-10 µM), were added at the time of wounding, and the rate of epithelial repair was measured in control and KGF-treated cells.

Statistics. Data are presented as means ± SE. Statistical analysis was done by unpaired Student's t-test or ANOVA, where appropriate. Significant differences found by ANOVA were further analyzed by Dunn's test for multiple comparisons. The results were considered statistically significant at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of KGF on the number of alveolar epithelial cells isolated. KGF (5 mg/kg) treatment significantly increased the number of alveolar epithelial cells isolated by elastase digestion (48 ± 5 × 106 vs. 26 ± 2 × 106, n = 9 independent experiments, P = 0.001).

Effect of KGF on in vitro rate of alveolar epithelial repair. KGF treatment increased the rate of epithelial repair by 96 ± 22% compared with control cells at 12 h (n = 9 independent experiments, P < 0.001; Fig. 1, A and B). This effect was not related to proliferation, since equal numbers of KGF-treated and control cells were plated and KGF-treated cells did not have enhanced proliferation in vitro (see below). The rate of repair measured at 24 h was also significantly increased (data not shown).


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Fig. 1.   Effect of in vivo keratinocyte growth factor (KGF) administration on the rate of in vitro alveolar epithelial repair. Alveolar epithelial cells were grown to confluent monolayers, and a mechanical wound was made using a pipette tip. A: rate of epithelial repair measured at 12 h was significantly greater in KGF-pretreated cells. Data are expressed as means ± SE; n = 9. * P < 0.001 compared with control. B: time lapse photograph of control monolayer compared with KGF-treated monolayer. Arrows indicate the wound edge. Note that by 24 h the KGF-treated monolayer has achieved near complete wound closure, whereas the control monolayer has not.

Effect of KGF on cell adhesion. KGF-treated cells were significantly more adherent than control cells to polystyrene, fibronectin, collagen type I, collagen type III, and collagen type IV at 20 h (Fig. 2). Increased adhesion to polystyrene was first evident at 8 h; however, by 48 h, there was no difference in adhesion between KGF-treated cells and control cells (data not shown), indicating that differential adhesion did not lead to a difference in cell number at the time of the alveolar epithelial repair assay.


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Fig. 2.   Effect of in vivo KGF administration on in vitro adherence to polystyrene and extracellular matrix proteins. Freshly isolated alveolar epithelial cells were plated (50,000 cells/well) on 96-well plates precoated with 1% BSA, fibronectin (10 µg/ml), collagen I, III, and IV (100 µg/ml), or without precoating (polystyrene). Cells were fixed and stained 20 h after plating, and absorbance was measured. Data are expressed as means ± SE; n = 4. * P = 0.01 and <0.001, 0.01, 0.01, and 0.01, respectively, KGF-treated cells vs. control cells.

Effect of KGF on cell spreading. In confluent monolayers, the change in internuclear distance between adjacent cells can be used to quantify the amount of cell spreading in response to a stimulus such as mechanical wounding. KGF-treated cells had significantly more cell spreading at the wound edge, as measured by internuclear distance, than control cells at the wound edge. KGF had no effect on the internuclear distance measured in the intact monolayer away from the wound edge (Fig. 3).


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Fig. 3.   Effect of in vivo KGF administration on cell spreading. Internuclear distance between cells on the same side of the wound edge and within the intact monolayer away from the wound edge was measured in KGF-treated and control cells. Data are expressed as means ± SE; n = 5. * P < 0.001, internuclear distance at the wound edge of KGF-treated cells vs. control cells.

Effect of in vivo administration of KGF on in vitro proliferation. No significant difference was found in in vitro proliferation between KGF-treated and control cells. In the tetrazolium-based proliferation assay, there was no difference in detected metabolic activity (control 1.3 ± 0.2 vs. KGF 1.2 ± 0.1 absorbance units, P = 0.5). Cell counts obtained 48 h after in vitro culture were similar (control 160 ± 24 × 103 vs. KGF 160 ± 24 × 103, P = 0.9).

Contribution of cell proliferation to the rate of epithelial repair of KGF-treated cells. There were no differences in BrDU incorporation at the wound edge or in the intact monolayer away from the wound edge in KGF-treated vs. control cells. In both groups, BrDU incorporation was minimal in the intact monolayer away from the wound edge (data not shown) and absent in cells at the wound edge (<1 BrDU-positive cells per high-power field at the wound edge during the epithelial repair assay).

Effect of supernatant from KGF-treated and control cells on the rate of alveolar epithelial repair. The supernatant collected from the KGF-treated cells at the end of the repair assay increased the rate of alveolar epithelial repair by 40 ± 13% when applied to untreated cells compared with the supernatant from control cells (P = 0.03, n = 5; Fig. 4). This effect was mediated, in part, by increased cell spreading at the wound edge, as was seen in the prior experiments. The internuclear distance between adjacent cells at the wound edge was 183 ± 14 µm compared with 159 ± 20 µm at the wound edge in the control (P < 0.001, n = 3).


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Fig. 4.   Effect of supernatant collected from KGF-treated and control cells after the epithelial repair assay on control cell alveolar epithelial repair. At the conclusion of the epithelial repair assay, supernatants were collected from KGF-treated and control cells and used as medium for control cell alveolar epithelial repair assays. Control cells treated with supernatant from KGF-treated cells had an increased rate of alveolar epithelial repair compared with control cells treated with control supernatant. Data are expressed as means ± SE; n = 5. * P = 0.03.

Effect of EGF receptor inhibitors on the epithelial repair capacity. Because the supernatant studies suggested that at least part of the KGF effect was mediated through release of a soluble mediator, and some of our prior studies indicated that alveolar epithelial repair can be enhanced through the activation of the EGF receptor pathway (18, 21), we hypothesized that release of TGF-alpha and/or EGF might mediate the enhanced epithelial repair. To assess the role of the TGF-alpha /EGF pathway in the enhanced epithelial repair rate observed after KGF treatment, AG-1478 and compound 56, both specific EGF receptor tyrosine kinase inhibitors (9, 15), were added in vitro at the time of monolayer wounding. Both inhibitors had significant, dose-dependent inhibitory effects on KGF-treated cells with no significant inhibitory effect on control cells (Fig. 5, A and B).


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Fig. 5.   Effect of epidermal growth factor (EGF) receptor blockade on the alveolar epithelial repair rate of KGF-treated cells. Tyrphostin AG-1478 (A) and compound 56 (B), both specific inhibitors of EGF receptor autophosphorylation (9, 15), inhibited KGF-augmented epithelial repair in a dose-dependent manner with no significant inhibitory effect on control cells. Data are expressed as means ± SE; n = 3 for AG-1478 and n = 4 for compound 56. * P < 0.05 and ** P < 0.01.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Acute lung injury is characterized by damage to the alveolar epithelium and endothelium with accumulation of plasma proteins in the alveoli leading to hypoxemic respiratory failure (2, 4, 5). The injured alveolar compartment is the focus of an intense inflammatory reaction, with the interaction of multiple growth factors and cytokines determining the ultimate resolution of lung injury (14, 26-28). Successful recovery requires the proliferation and differentiation of alveolar epithelial type II cells to reconstitute epithelial integrity (1, 4). KGF is a potent epithelial cell mitogen that attenuates experimentally induced lung injury when given before injury (11, 23, 44, 46). The simplest explanation for the protective effect of KGF is enhanced proliferation of alveolar epithelial type II cells. However, alveolar type II cell proliferation is unlikely to be the sole mechanism by which KGF attenuates lung injury. Several recent studies have shown that KGF has multiple nonmitogenic effects. For example, KGF increases expression of surfactant proteins (33, 45), enhances alveolar fluid clearance in part by nonmitogenic mechanisms (36), and increases expression of the Na+-K+-ATPase by alveolar epithelial type II cells (8).

Although a series of experimental lung injury studies in rats has shown that treatment with KGF prevents lung injury and/or hastens recovery from injury (11, 19, 23, 25, 34, 39, 43, 44), the design of those studies did not allow for separation of the mitogenic effect of KGF from other beneficial effects. Therefore, the main objective of this study was to assess nonproliferative mechanisms by which KGF might ameliorate lung injury in vivo. To achieve this objective, the dose, timing, and route of KGF administration were identical to those used in studies of experimental lung injury. However, the number of cells used in the in vitro assays was equal in the KGF and control arm. This allowed for analysis of the rate of alveolar epithelial repair of KGF-treated cells while eliminating the mitogenic effect. The primary hypothesis was that in vivo KGF treatment would enhance the rate of alveolar epithelial repair of type II cells in an in vitro model.

A single dose of KGF in vivo resulted in a 96 ± 22% increase in the rate of epithelial repair in vitro, a remarkable effect that was not the result of enhanced cell proliferation. Thus KGF treatment alters basic biological properties of alveolar epithelial cells and stimulates proliferation in vivo. Furthermore, the effect of a single dose of KGF on alveolar epithelial cells persists 4 or 5 days after administration.

Because KGF treatment increased the rate of epithelial repair, we assessed differences in the ability of KGF-treated cells to adhere, spread, and proliferate in vitro. Enhanced epithelial cell adherence could protect against lung injury by providing a better physical barrier in the face of noxious pathological stimuli. After lung injury has been sustained, the ability to adhere to extracellular matrix proteins deposited on the denuded basement membrane may allow for more rapid restoration of the alveolar architecture. KGF treatment in vitro increases keratinocyte adhesion to extracellular matrix proteins (29). In the current study, KGF-treated alveolar epithelial type II cells were more adherent to polystyrene and extracellular matrix proteins in the first 10-24 h. By 48 h, the time of the epithelial repair assay, there was no longer any difference in adherence to polystyrene, indicating that differential adherence did not lead to a difference in cell number at the time of the alveolar epithelial repair assay.

Cell spreading and migration are early mechanisms of repair that are contingent on adhesion followed by deadhesion to the extracellular matrix (10, 30, 32). Enhanced cell spreading with KGF treatment was a major mechanism by which KGF increased the rate of alveolar epithelial repair. Cell migration likely played a similar role.

Cell proliferation is an important consequence of in vivo treatment with KGF (13, 22). However, alveolar epithelial type II cells have a low proliferative index when cultured in vitro (25, 35, 36). As expected, KGF treatment was a potent in vivo mitogen, doubling the number of alveolar epithelial cells that were isolated. However, this mitogenic effect did not persist in culture. Proliferation during culture for 48 h and during the epithelial repair assay was minimal and not significantly different in KGF-treated and control cells. The lack of in vitro proliferation after KGF treatment supports the hypothesis that KGF can enhance the rate of epithelial repair of alveolar epithelial type II cells independent of its mitogenic effect.

KGF is of important clinical interest because, in experimental models, it favors the maintenance and/or restoration of normal lung architecture in the face of injury. The ultimate effect of KGF in vivo is likely influenced by its effects on other mediators of epithelial repair. For example, we have recently found that interleukin (IL)-1beta , a cytokine found in elevated concentrations in the pulmonary edema fluid of patients with acute lung injury, promotes in vitro alveolar epithelial repair through an EGF/TGF-alpha mechanism (18). Previous work in keratinocytes demonstrated that KGF induces cell proliferation by increasing TGF-alpha mRNA and protein levels, with subsequent autophosphorylation and activation of the EGF receptor (12). Stimulation of intestinal epithelial cells by KGF results in EGF receptor autophosphorylation and an increase in TGF-alpha precursor proteins (6). Therefore, we tested the hypothesis that the enhanced epithelial repair rate of alveolar epithelial cells after KGF treatment involves signaling through the EGF receptor. In fact, EGF receptor blockade by two specific inhibitors effectively negated the effect of KGF with no significant effect on control cells. These results suggest the effects of KGF on alveolar epithelial cells are mediated, in part, by the EGF receptor pathway. These findings, coupled with our previous work with IL-1beta (18) and TGF-alpha (21), indicate that the EGF receptor may serve as a final common pathway to stimulate alveolar epithelial repair in vitro.

In summary, this study demonstrates that a single in vivo intratracheal administration of KGF to rats 48 h before alveolar epithelial cell isolation results in enhanced in vitro alveolar epithelial repair. The augmented rate of epithelial repair is the result of enhanced cell spreading and migration, but not cell proliferation, and is, in part, dependent on the EGF receptor pathway. These results provide several novel insights into the nonmitogenic effects of KGF on alveolar epithelial cells, which may be important in alveolar epithelial repair, and help to explain why KGF treatment ameliorates experimental acute lung injury.


    ACKNOWLEDGEMENTS

This study was supported by National Heart, Lung, and Blood Institute Grants HL-51856 and HL-51854.


    FOOTNOTES

Address for reprint requests and other correspondence: M. A. Matthay, Cardiovascular Research Institute, Univ. of California, San Francisco, CA 94143-0130 (E-mail: mmatt{at}itsa.ucsf.edu).

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.

First published February 22, 2002;10.1152/ajplung.00396.2001

Received 11 October 2001; accepted in final form 12 February 2002.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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