Bronchial Epithelial Cells Produce Chemotactic Activity for Bronchial Epithelial Cells Possible Role for Fibronectin in Airway Repair1,2
SHUNSUKE SHOJI, RONALD F. ERTL, JAMES LINDER, DEBRA J. ROMBERGER, and STEPHEN I. RENNARD
Introduction
The epithelial cells lining the surface of the airway are frequently exposed to environmental toxins and pathogens that cause airway injury. They are also damaged during conditions such as asthma, bronchitis, and pneumonia (1).These injuries of the airway epithelial surface can result in sloughing of epithelial cells, resulting in a partially denuded basement membrane. Although the mechanisms that lead to repair after injury of the airway are not fully known, the restoration of normal airway function and architecture requires a prompt and orderly repair of any epithelial defect. As a number of studies have demonstrated that different types of epithelial cells (2-4), including bronchial epithelial cells (5, 6), are capable of chemotactic migration to specific stimuli, we hypothesized that bronchial epithelial cellsthat survive desquamation after airway injury might recruit other bronchial epithelial cells from the margin of the injured area. We evaluated the capacity ofcultured airway epithelial cells to release chemotactic activity for airway epithelial cells. The current study demonstrates that bronchial epithelial cells release a chemotactic factor for airway epithelial cells, and it appears to be fibronectin. This cellular fibronectin produced by bronchial epithelial cells, moreover, appears to have increased potency as a chemoattractant for bronchial epithelial cells compared with bovine plasma fibronectin and may, therefore, have a role during reepithelialization after bronchial injury. Methods Preparation of Bronchial Epithelial Cells Bronchial epithelial cells were obtained by a modification of the method of Wu and Smith (7). Briefly, bovine lungs were obtained from a slaughterhouse immediately after the ani218
SUMMARY Bronchial mucosal Injury Initiates a complex series of repair mechanisms, one of which Is reeplthellallzatlon of a denuded lumenal surface. This suggests the hypothesis that bronchial epithelial cells, the cells Initially affected by bronchial Injury, might be able to Initiate repair of an Injured area by producing a chemotectlc activity for Intect bronchial epithelial cells. To evaluate this, bronchial epithelial cells _re prepared from bovine lung by protease digestion and cultured In medium 199 (M199)with 10% fetel calf serum (FCS) until confluence, after which the cells _re rinsed with Hanks' balanced selt solution, and serum-free fresh M199 _s added. This conditioned medium _s then collected and used to test the chemotectlc response of bronchial epithelial cells using a blind_II chamber technique. Target cells for this assay were Isolated from airways by protease digestion, grown to confluence In M199with 10% FCS,and then harveated with trypsin. Bronchial epithelial ceil-conditioned medium harveated after 3 days attracted more cells (197.0 ± 5.7 cells/10 high power fields) than did Ml99 without FCS alone (4.3 ± 0.9) (p < 0.01). Check8rboard analysis showed that the migration _s chemotectlc. The chemotactic activity _s nondlalyzable, pepsln.lablle, acld·atable, heat-labile, and IIpld-lnextraeteble. The chemotectlc activity accumulated In the culture medium with time. The addition of 25/1g1ml of cycloheximide Inhibited this accumulation. Column chromatography with Sephadex G·l50 revealed a single peak of chemoteetlc activity In the high molecular _Ight range. The chemotactic activity _s bound to gelatln-Sepharose 4B and _s eluted with 6 M urea. Enzyme-linked Immunosorbent assay of the column fractions demonatrated that the chemotectlc activity coeluted with flbronectln. In addition, antlflbronectln antibody caused Inhibition of the chemotectlc activity, strongly suggesting that the chemotectlc activity may be flbroneetln. Finally, bronchial epithelial ceil-derived flbroneetln was compared with plume-derlved flbronectln and found to be 10-to 50-fold more potent as a chemotectlc fector. The ability of bronchial epithelial cells to produce chemotectlc activity may play an Important role during tissue repair after airway InjUry by Initiating prompt and efficient reeplthellallzatlon of bronchial wall. AM REV RESPIR DIS 1990; 141:218-225
mal was killed. The lungs were immediately immersed in Leibovitz 15 medium (GIBCO, Grand Island, NY), and the bronchi were removed, cut into large pieces, and trimmed of connective tissue. The bronchi were then put into Eagle minimum essential medium (MEM) (GIBCO), which contained 0.1070 bacterial protease (type XIV; Sigma Chemical, St. Louis, MO) and incubated at 4 0 C overnight. The bronchial lumen was washed with MEM containing 10% fetal calf serum (FCS) (Biofluids, Rockville, MD) to detach the bronchial epithelial cells. The bronchial epithelial cells were then washed once with MEM with 10% FCS, filtered through l00-l1m nitex mesh (Tetko, Elmsford, NY), and resuspended in medium 199 (MI99) (GIBCO) supplemented with epidermal growth factor (25 ng/ml; Sigma), hydrocortisone (l ug/ml; Sigma), transferrin (5 ug/rnl; Sigma), insulin (5 ug/ml; Sigma), gentamicin (60 ug/ml; Elkins-Sinn, Cherry
Hill, NJ), penicillin-streptomycin (50 ug/rnl; Sigma), fungizone (2I1g/mI;Sigma), and 10% FCS at 1 x 106 cells/mI. The cells were plated in tissue culture dishes 100mm in diameter (Corning, Corning, NY), 10 ml per dish. The cells were then incubated at 370 C in 5% CO 2 for three days to allow the cells to
(Received in original form April 3, 1989 and in revised form June 3, 1989) I From the Pulmonary and Critical Care Medicine Section, Department of Internal Medicine and Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska. 2 Correspondence and requests for reprints should be addressed to Stephen I. Rennard, M.D., Pulmonary Section, Department of Internal Medicine, University of Nebraska Medical Center, 42nd and Dewey Avenue, Omaha, NE 68105.
AIRWAY EPITHELIAL CELL-DIRECTED EPITHELIAL CHEMOTAXIS
attach and grow. Under these conditions, the cells grew to near confluence in three days. In order to obtain bronchial epithelial cellconditioned medium, confluent monolayers of bronchial epithelial cellswere washed twice with Hanks' balanced salt solution (HBSS) (GIBCO) to remove serum, and the medium was changed to M199 with antibiotics, but without other supplements. The cells wereincubated at 37 0 C in 5% CO 2 for 3 more days, and the epithelial cell supernatant-conditioned medium was harvested. In order to obtain bronchial epithelial cells as the cell target for chemotaxis, bronchial epithelial cell monolayers were washed twice with HBSS and 0.25070 trypsin (GIBCO) with 0.02% disodium ethylenediaminetetraacetic acid (EDTA)(Fisher, St. Louis, MO) was added to detach the cells. The detached cells were centrifuged once with MEM containing 10% FCS to neutralize trypsin and washed twice with HBSS to remove serum and resuspended at 1 x 106 cells/ml in M199 without other supplements. These cellsweremore than 95% viable by trypan blue exclusion.
Bronchial Epithelial Cell Chemotaxis Bronchial epithelial cellchemotaxis was measured by the Boyden's blindwell chamber (Neuroprobe, Bethesda, MD) (8). An B-urn pore size filter membrane' (Nuclopore, Pleasanton, CA) coated with 0.1% gelatin (Bio-Rad, Richmond, CA) was used. Gelatincoated filters wereused following the methods of Postlethwaite and coworkers (9) and Blay and Brown (10). Uncoated filters did not support chemotaxis, presumably because of inadequate cellular attachment. The conditioned medium from bronchial epithelial cells was placed into each of the bottom wells as a chemoattractant. Bronchial epithelial cells prepared as described above were placed into each of the top wellsof the chemotaxis chamber. The chambers were then incubated at 37 0 C in 5% CO 2 for 6 h. After incubation, cells on the top of the filter were removed by scraping. The filter was then stained with a modified Wright stain (LeukoStat; Fisher). Epithelial cell chemotaxis activity is shown as the total number of migrated epithelial cells counted in 10 high power fields using a light microscope (Olympus, Lake Success, NY) at a magnification x 400. Selected membranes were stained by antikeratin antibody (MAK-6; Triton, Alameda, CA) and antivimentin antibody (DAKO-Vimentin; DAKO, Santa Barbara, CA) to confirm that the migrating cells wereof epithelial origin and that there was no contamination of mesenchymal cells, especially fibroblasts. Checkerboard Analysis Toevaluate that cell migration was chemotactic, checkerboard analysis was performed (11). In the top wells of the chemotactic chamber, various dilutions of medium conditioned with bovine bronchial epithelial cells (1:1, 1:2, 1:4, 0) were applied. In the bottom wells, the same concentrations of bronchial epithelial cell-
219
conditioned medium wereplaced such that all combinations of concentrations were tested. Each combination was assayed in triplicate.
ous phase was also dried to remove any remaining ethyl acetate. The dried aqueous phase was then reconstituted with MI99.
Dose Response of Epithelial Migration The dose response wasmeasured by using variously diluted concentrations of medium conditioned with bovine bronchial epithelial cell supernatants as chemoattractants. To confirm that the increase of migrated epithelial cells was not due to increased cell attachment to the chemotaxis membrane, the dose response was also measured with another 48-wellchamber with the same chemoattractants to which epithelial cellshad been "preattached." This was achieved by incubating epithelial cells at I x 106 cells/ml in the top wells and M199 in the bottom wells for two hours. After allowing attachment, the filter was transferred very carefully to a new chemotactic chamber. This second chamber, which contained chemoattractants in the bottom wells, was then incubated for 6 h, and the migrated cells were counted.
Partial Purification of the Chemotactic Activity by Column Chromatography In order to determine the apparent molecular weightof the activity,the medium conditioned with bovine bronchial epithelial cells (2 ml) was applied to a Sephadex G-150column (1.6 x 116cm; Pharmacia, Piscataway, NJ), and bronchial epithelialcellchemotaxis was assayed for each fraction. Blue dextran (2 million daltons), bovine serum albumin (66,000daltons), and phenol red (354daltons) wereused as molecular weight markers. To determine if the chemotactic activity was distinct from fibronectin, a gelatin-Sepharose 4B column chromatography (pharmacia) was used to remove gelatin-binding proteins, including fibronectin, which was eluted with 6 M urea. Each of the above fractions was dialyzed against culture medium, and the chemotactic activity was determined as described above. The fibronectin concentration in each of the fractions was determined by enzyme-linkedimmunosorbent assay (ELISA) (12).
Time-dependent and De Novo Production of Chemotactic Activity by Bronchial Epithelial Cells In order to determine the time dependence of the release of chemotactic activity by bovine bronchial epithelial cells, confluent cultures of bovine bronchial epithelial cells prepared as described above were incubated in M199 without serum, and aliquots were taken after various times of incubation. In parallel cultures, 25 ug/ml of cycloheximide (Sigma) were added at the beginning of the culture. Harvested media (2 ml) were dialyzed against M199 (1,000 ml) with two exchanges of M199 to remove cycloheximide. The chemotactic activity present was assayed as described above. Partial Characterization of Chemotactic Activity In order to partially characterize the chemotactic activity in the bovine epithelial cell-conditioned medium, a variety of techniques was used. First, dialysis was done (Spectrum, Los Angeles, CA; molecular weight cutoff, 12,000 to 14,000)against MI99 at room temperature for 6 h with two exchanges of medium. Sensitivity to proteases was assessed by pepsin digestion, which was done by incubation of the conditioned medium with 0.1 mg/ml pepsin (Sigma) for 2 h at 37 0 C after adjusting the pH to 2.5 with 0.5 M acetic acid. After digestion, the pH was adjusted to 7.4 with TRIS base, and the sample was dialyzed against M199. Sensitivity to acid was done by incubation with 0.5 M acetic acid at pH 2.5 and 37 0 C for 2 h, after which the sample was neutralized and dialyzed against M199. Sensitivity to heat was done by boiling the sample for 15min. The lipid extractability of the activity was done by vortexing the sample with two volumes of ethyl acetate twice. The lipid phase was removed, dried under nitrogen, and resuspended in MI99. The aque-
Inhibition of Partially Purified Chemotactic Activity by Antifibronectin Antiserum To confirm that the peak of the chemotactic activity in gelatin-binding portion is fibronectin, the peak fraction (4 ml) from gelatinbinding portion was dialyzed against MI99 (700 ml) for 6 h, with two exchanges of M199 to remove urea, and applied to bottom wells of the chemotactic chamber with and without 1:10diluted antibovine plasma fibronectin antiserum (13) in M199, and bronchial epithelial cellchemotaxis was measured as described above; 100 ug/ml of porcine pancreatic insulin (Sigma) were used as a positive control for chemotaxis (5), and MI99 was used as a negative control. Toexclude the possibility of nonspecific inhibition of chemotaxis by antiserum, normal rabbit serum diluted at 1:10in M199 was applied as a control. Comparison of the Potency of Fibronectins from Different Sources as the Chemoattractants for Bronchial Epithelial Cells Because a previous report has indicated that cellular fibronectin is more potent than plasma fibronectin as a chemoattractant for fibroblast (14), there also may be functional differences between plasma fibronectin and the fibronectin locally produced by bronchial epithelial cells. Therefore, we purified the fibronectin from bovine plasma and from conditioned media from bovine bronchial epithelial cells using gelatin-Sepharose fractionation followed by dialysis against M199. The fibronectin preparations were diluted serially with M199, and their chemotactic activity for bronchial epithelial cells was measured. The concentration of fibronectin in the samples was measured by ELISA (12).
SHOJI, ERTL, LINDER, ROMBERGER, AND RENNARD
220
Results
Bronchial Epithelial Cell Chemotaxis Bronchial epithelial cells demonstrated chemotactic migration in response to bronchial epithelial cell-conditioned medium. The number of cellsinduced to migrate was significantly greater in response to conditioned medium (197.0 ± 5.7 cells/l0 high power fields)that in response to control medium (4.3 ± 0.9 cells/l0 high power fields) (p < 0.01). Selected membranes were stained by antikeratin antibody and antivimentin antibody to confirm that the migrating cells wereof epithelial origin. The results revealed most of the cells (more than 95%) were keratin-positive and vimentin-negative, thus indicating that the responding cells were epithelial (figure 1). Checkerboard Analysis Checkerboard analysis demonstrated that the cell migration was due to both chemotaxis (directed migration) and chemokinesis (random migration) (table 1). The cell migration was measured in the presence of various dilutions ofmedium conditioned with bovine bronchial epithelial cells both above and below the filter membrane in order to make gradients of chemoattractant. Chemokinesis was present because even when no gradient existed, the number of cells that migrated increased as the concentration of conditioned medium increased. The cell migration was also chemotactic, however, because the number of cellsthat migrated increased when a gradient was present. Thus, bronchial epithelial cell migration in response to bronchial epithelial cell-conditioned medium was both chemokinetic and chemotactic. Dose Response of Epithelial Cell Migration The number of migrated epithelial cells decreased when the bronchial epithelial cell-conditioned medium was diluted with culture medium, indicating a dose dependence of the chemotactic activity. Cell migration with a chemotaxis membrane to which cellshad been preattached showed a similar dose dependence, indicating that the epithelial cell migration was not due to increased cell attachment (figure 2). Time-dependent and De Novo Production of Chemotactic Activity by Bovine Bronchial Epithelial Cells Bovine bronchial epithelial cells accumulated chemotactic activity in the supernatant medium with increasing time,
Fig. 1. Bronchial epithelial cells migrated through the chemotactic membrane. Top panel. Migrated bronchial epithelial cells stained by LeukoStat. Middlepanel. Migrated cells stained by antikeratin antibody. Bottom panel. Migrated cells stained by antivimentin antibody; magnification: x 400.
221
AIRWAY EPITHELIAL CELL-DIRECTED EPITHELIAL CHEMOTAXIS
TABLE 1 CHECKERBOARD ANALYSIS OF CELL MIGRATION Dilution' Above Membranet
o Dilution below membrane:!:
o
3.0 3.3 10.7 62.0
1:4 1:2 1:1
± 0.6 ± 0.3 ± 0.7 ± 5.3
2.0 3.3 13.0 62.7
± 0.6 ± 0.3 ± 3.1 ± 0.7
1.7 1.7 8.3 44.7
± 0.3 ± 0.3 ± 2.3 ± 3.0
1.3 1.7 7.0 39.3
± 0.3 ± 0.7 ± 2.0 ± 2.3
, Ratio of dilution of crude bronchial epithelial cell-condilioned medium. 8ronchial epithelial cell suspension at 1 )( 10' cellslml in diluted conditioned medium. Diluted conditioned medium as a chemoattractant (number of migrated cells is indicated as average In triplicate ± SEM).
t
*
reaching a plateau at about three days. The addition of cycloheximide (25 ug/ ml) at the beginning of the incubation inhibited the release of chemotactic activity from bovine bronchial epithelial cells (figure 3). This suggests that the accumulation of chemotactic activity might represent de novo synthesis.
Partial Characterization of Chemotactic Activity The partial characterization of chemotactic activity in the medium conditioned with bovine bronchial epithelial cells revealed that the activity was nondialyza-
LL
ble, pepsin-labile, acid-stable, heat-labile, and lipid-inextractable (figure 4).
Inhibition of the Chemotactic Activity by Antifibronectin Antiserum Demonstration ofthe Molecular Size by The chemotactic activity of the gelatin0-150 Molecular Sieve Column binding portion was significantly inhibitChromatography ed by antifibronectin antiserum, and not To demonstrate the approximate molec- by normal rabbit serum. This confirms ular size of bovine bronchial epithelial that the gelatin-binding portion of checell-derived chemotactic activity, Sepha- motactic activity is fibronectin. Insulindex G-150 column chromatography was directed bronchial epithelial cell cheused. Chemotactic activity eluted as a sin- motaxis (5), however, was not inhibited gle sharp peak near the void volume of by antifibronectin antiserum, indicating the column. This peak coincided with the that inhibition of chemotaxis was not peak of fibronectin, which was measured a nonspecific effect of antifibronectin by ELISA (figure 5). antiserum (figure 7). Comparison of the Potency of Fibronectins from Different Sources as the Chemoattractants for Bronchial Epithelial Cells The chemotaxis assay using serially diluted fibronectins purified by gelatin-Sepharose 4B column chromatography from both bovine plasma and bovine epithelial cell-conditioned medium showed that locally produced fibronectin is more potent than plasma fibronectin as a chemoattractant. The difference is approximately 10- to 50-fold (figure 8).
100
Q.
J: 0 ~
......
en
..J ..J W
0
..J
SHUNSUKE SHOJI, RONALD F. ERTL, JAMES LINDER, DEBRA J. ROMBERGER, and STEPHEN I. RENNARD
Introduction
The epithelial cells lining the surface of the airway are frequently exposed to environmental toxins and pathogens that cause airway injury. They are also damaged during conditions such as asthma, bronchitis, and pneumonia (1).These injuries of the airway epithelial surface can result in sloughing of epithelial cells, resulting in a partially denuded basement membrane. Although the mechanisms that lead to repair after injury of the airway are not fully known, the restoration of normal airway function and architecture requires a prompt and orderly repair of any epithelial defect. As a number of studies have demonstrated that different types of epithelial cells (2-4), including bronchial epithelial cells (5, 6), are capable of chemotactic migration to specific stimuli, we hypothesized that bronchial epithelial cellsthat survive desquamation after airway injury might recruit other bronchial epithelial cells from the margin of the injured area. We evaluated the capacity ofcultured airway epithelial cells to release chemotactic activity for airway epithelial cells. The current study demonstrates that bronchial epithelial cells release a chemotactic factor for airway epithelial cells, and it appears to be fibronectin. This cellular fibronectin produced by bronchial epithelial cells, moreover, appears to have increased potency as a chemoattractant for bronchial epithelial cells compared with bovine plasma fibronectin and may, therefore, have a role during reepithelialization after bronchial injury. Methods Preparation of Bronchial Epithelial Cells Bronchial epithelial cells were obtained by a modification of the method of Wu and Smith (7). Briefly, bovine lungs were obtained from a slaughterhouse immediately after the ani218
SUMMARY Bronchial mucosal Injury Initiates a complex series of repair mechanisms, one of which Is reeplthellallzatlon of a denuded lumenal surface. This suggests the hypothesis that bronchial epithelial cells, the cells Initially affected by bronchial Injury, might be able to Initiate repair of an Injured area by producing a chemotectlc activity for Intect bronchial epithelial cells. To evaluate this, bronchial epithelial cells _re prepared from bovine lung by protease digestion and cultured In medium 199 (M199)with 10% fetel calf serum (FCS) until confluence, after which the cells _re rinsed with Hanks' balanced selt solution, and serum-free fresh M199 _s added. This conditioned medium _s then collected and used to test the chemotectlc response of bronchial epithelial cells using a blind_II chamber technique. Target cells for this assay were Isolated from airways by protease digestion, grown to confluence In M199with 10% FCS,and then harveated with trypsin. Bronchial epithelial ceil-conditioned medium harveated after 3 days attracted more cells (197.0 ± 5.7 cells/10 high power fields) than did Ml99 without FCS alone (4.3 ± 0.9) (p < 0.01). Check8rboard analysis showed that the migration _s chemotectlc. The chemotactic activity _s nondlalyzable, pepsln.lablle, acld·atable, heat-labile, and IIpld-lnextraeteble. The chemotectlc activity accumulated In the culture medium with time. The addition of 25/1g1ml of cycloheximide Inhibited this accumulation. Column chromatography with Sephadex G·l50 revealed a single peak of chemoteetlc activity In the high molecular _Ight range. The chemotactic activity _s bound to gelatln-Sepharose 4B and _s eluted with 6 M urea. Enzyme-linked Immunosorbent assay of the column fractions demonatrated that the chemotectlc activity coeluted with flbronectln. In addition, antlflbronectln antibody caused Inhibition of the chemotectlc activity, strongly suggesting that the chemotectlc activity may be flbroneetln. Finally, bronchial epithelial ceil-derived flbroneetln was compared with plume-derlved flbronectln and found to be 10-to 50-fold more potent as a chemotectlc fector. The ability of bronchial epithelial cells to produce chemotectlc activity may play an Important role during tissue repair after airway InjUry by Initiating prompt and efficient reeplthellallzatlon of bronchial wall. AM REV RESPIR DIS 1990; 141:218-225
mal was killed. The lungs were immediately immersed in Leibovitz 15 medium (GIBCO, Grand Island, NY), and the bronchi were removed, cut into large pieces, and trimmed of connective tissue. The bronchi were then put into Eagle minimum essential medium (MEM) (GIBCO), which contained 0.1070 bacterial protease (type XIV; Sigma Chemical, St. Louis, MO) and incubated at 4 0 C overnight. The bronchial lumen was washed with MEM containing 10% fetal calf serum (FCS) (Biofluids, Rockville, MD) to detach the bronchial epithelial cells. The bronchial epithelial cells were then washed once with MEM with 10% FCS, filtered through l00-l1m nitex mesh (Tetko, Elmsford, NY), and resuspended in medium 199 (MI99) (GIBCO) supplemented with epidermal growth factor (25 ng/ml; Sigma), hydrocortisone (l ug/ml; Sigma), transferrin (5 ug/rnl; Sigma), insulin (5 ug/ml; Sigma), gentamicin (60 ug/ml; Elkins-Sinn, Cherry
Hill, NJ), penicillin-streptomycin (50 ug/rnl; Sigma), fungizone (2I1g/mI;Sigma), and 10% FCS at 1 x 106 cells/mI. The cells were plated in tissue culture dishes 100mm in diameter (Corning, Corning, NY), 10 ml per dish. The cells were then incubated at 370 C in 5% CO 2 for three days to allow the cells to
(Received in original form April 3, 1989 and in revised form June 3, 1989) I From the Pulmonary and Critical Care Medicine Section, Department of Internal Medicine and Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska. 2 Correspondence and requests for reprints should be addressed to Stephen I. Rennard, M.D., Pulmonary Section, Department of Internal Medicine, University of Nebraska Medical Center, 42nd and Dewey Avenue, Omaha, NE 68105.
AIRWAY EPITHELIAL CELL-DIRECTED EPITHELIAL CHEMOTAXIS
attach and grow. Under these conditions, the cells grew to near confluence in three days. In order to obtain bronchial epithelial cellconditioned medium, confluent monolayers of bronchial epithelial cellswere washed twice with Hanks' balanced salt solution (HBSS) (GIBCO) to remove serum, and the medium was changed to M199 with antibiotics, but without other supplements. The cells wereincubated at 37 0 C in 5% CO 2 for 3 more days, and the epithelial cell supernatant-conditioned medium was harvested. In order to obtain bronchial epithelial cells as the cell target for chemotaxis, bronchial epithelial cell monolayers were washed twice with HBSS and 0.25070 trypsin (GIBCO) with 0.02% disodium ethylenediaminetetraacetic acid (EDTA)(Fisher, St. Louis, MO) was added to detach the cells. The detached cells were centrifuged once with MEM containing 10% FCS to neutralize trypsin and washed twice with HBSS to remove serum and resuspended at 1 x 106 cells/ml in M199 without other supplements. These cellsweremore than 95% viable by trypan blue exclusion.
Bronchial Epithelial Cell Chemotaxis Bronchial epithelial cellchemotaxis was measured by the Boyden's blindwell chamber (Neuroprobe, Bethesda, MD) (8). An B-urn pore size filter membrane' (Nuclopore, Pleasanton, CA) coated with 0.1% gelatin (Bio-Rad, Richmond, CA) was used. Gelatincoated filters wereused following the methods of Postlethwaite and coworkers (9) and Blay and Brown (10). Uncoated filters did not support chemotaxis, presumably because of inadequate cellular attachment. The conditioned medium from bronchial epithelial cells was placed into each of the bottom wells as a chemoattractant. Bronchial epithelial cells prepared as described above were placed into each of the top wellsof the chemotaxis chamber. The chambers were then incubated at 37 0 C in 5% CO 2 for 6 h. After incubation, cells on the top of the filter were removed by scraping. The filter was then stained with a modified Wright stain (LeukoStat; Fisher). Epithelial cell chemotaxis activity is shown as the total number of migrated epithelial cells counted in 10 high power fields using a light microscope (Olympus, Lake Success, NY) at a magnification x 400. Selected membranes were stained by antikeratin antibody (MAK-6; Triton, Alameda, CA) and antivimentin antibody (DAKO-Vimentin; DAKO, Santa Barbara, CA) to confirm that the migrating cells wereof epithelial origin and that there was no contamination of mesenchymal cells, especially fibroblasts. Checkerboard Analysis Toevaluate that cell migration was chemotactic, checkerboard analysis was performed (11). In the top wells of the chemotactic chamber, various dilutions of medium conditioned with bovine bronchial epithelial cells (1:1, 1:2, 1:4, 0) were applied. In the bottom wells, the same concentrations of bronchial epithelial cell-
219
conditioned medium wereplaced such that all combinations of concentrations were tested. Each combination was assayed in triplicate.
ous phase was also dried to remove any remaining ethyl acetate. The dried aqueous phase was then reconstituted with MI99.
Dose Response of Epithelial Migration The dose response wasmeasured by using variously diluted concentrations of medium conditioned with bovine bronchial epithelial cell supernatants as chemoattractants. To confirm that the increase of migrated epithelial cells was not due to increased cell attachment to the chemotaxis membrane, the dose response was also measured with another 48-wellchamber with the same chemoattractants to which epithelial cellshad been "preattached." This was achieved by incubating epithelial cells at I x 106 cells/ml in the top wells and M199 in the bottom wells for two hours. After allowing attachment, the filter was transferred very carefully to a new chemotactic chamber. This second chamber, which contained chemoattractants in the bottom wells, was then incubated for 6 h, and the migrated cells were counted.
Partial Purification of the Chemotactic Activity by Column Chromatography In order to determine the apparent molecular weightof the activity,the medium conditioned with bovine bronchial epithelial cells (2 ml) was applied to a Sephadex G-150column (1.6 x 116cm; Pharmacia, Piscataway, NJ), and bronchial epithelialcellchemotaxis was assayed for each fraction. Blue dextran (2 million daltons), bovine serum albumin (66,000daltons), and phenol red (354daltons) wereused as molecular weight markers. To determine if the chemotactic activity was distinct from fibronectin, a gelatin-Sepharose 4B column chromatography (pharmacia) was used to remove gelatin-binding proteins, including fibronectin, which was eluted with 6 M urea. Each of the above fractions was dialyzed against culture medium, and the chemotactic activity was determined as described above. The fibronectin concentration in each of the fractions was determined by enzyme-linkedimmunosorbent assay (ELISA) (12).
Time-dependent and De Novo Production of Chemotactic Activity by Bronchial Epithelial Cells In order to determine the time dependence of the release of chemotactic activity by bovine bronchial epithelial cells, confluent cultures of bovine bronchial epithelial cells prepared as described above were incubated in M199 without serum, and aliquots were taken after various times of incubation. In parallel cultures, 25 ug/ml of cycloheximide (Sigma) were added at the beginning of the culture. Harvested media (2 ml) were dialyzed against M199 (1,000 ml) with two exchanges of M199 to remove cycloheximide. The chemotactic activity present was assayed as described above. Partial Characterization of Chemotactic Activity In order to partially characterize the chemotactic activity in the bovine epithelial cell-conditioned medium, a variety of techniques was used. First, dialysis was done (Spectrum, Los Angeles, CA; molecular weight cutoff, 12,000 to 14,000)against MI99 at room temperature for 6 h with two exchanges of medium. Sensitivity to proteases was assessed by pepsin digestion, which was done by incubation of the conditioned medium with 0.1 mg/ml pepsin (Sigma) for 2 h at 37 0 C after adjusting the pH to 2.5 with 0.5 M acetic acid. After digestion, the pH was adjusted to 7.4 with TRIS base, and the sample was dialyzed against M199. Sensitivity to acid was done by incubation with 0.5 M acetic acid at pH 2.5 and 37 0 C for 2 h, after which the sample was neutralized and dialyzed against M199. Sensitivity to heat was done by boiling the sample for 15min. The lipid extractability of the activity was done by vortexing the sample with two volumes of ethyl acetate twice. The lipid phase was removed, dried under nitrogen, and resuspended in MI99. The aque-
Inhibition of Partially Purified Chemotactic Activity by Antifibronectin Antiserum To confirm that the peak of the chemotactic activity in gelatin-binding portion is fibronectin, the peak fraction (4 ml) from gelatinbinding portion was dialyzed against MI99 (700 ml) for 6 h, with two exchanges of M199 to remove urea, and applied to bottom wells of the chemotactic chamber with and without 1:10diluted antibovine plasma fibronectin antiserum (13) in M199, and bronchial epithelial cellchemotaxis was measured as described above; 100 ug/ml of porcine pancreatic insulin (Sigma) were used as a positive control for chemotaxis (5), and MI99 was used as a negative control. Toexclude the possibility of nonspecific inhibition of chemotaxis by antiserum, normal rabbit serum diluted at 1:10in M199 was applied as a control. Comparison of the Potency of Fibronectins from Different Sources as the Chemoattractants for Bronchial Epithelial Cells Because a previous report has indicated that cellular fibronectin is more potent than plasma fibronectin as a chemoattractant for fibroblast (14), there also may be functional differences between plasma fibronectin and the fibronectin locally produced by bronchial epithelial cells. Therefore, we purified the fibronectin from bovine plasma and from conditioned media from bovine bronchial epithelial cells using gelatin-Sepharose fractionation followed by dialysis against M199. The fibronectin preparations were diluted serially with M199, and their chemotactic activity for bronchial epithelial cells was measured. The concentration of fibronectin in the samples was measured by ELISA (12).
SHOJI, ERTL, LINDER, ROMBERGER, AND RENNARD
220
Results
Bronchial Epithelial Cell Chemotaxis Bronchial epithelial cells demonstrated chemotactic migration in response to bronchial epithelial cell-conditioned medium. The number of cellsinduced to migrate was significantly greater in response to conditioned medium (197.0 ± 5.7 cells/l0 high power fields)that in response to control medium (4.3 ± 0.9 cells/l0 high power fields) (p < 0.01). Selected membranes were stained by antikeratin antibody and antivimentin antibody to confirm that the migrating cells wereof epithelial origin. The results revealed most of the cells (more than 95%) were keratin-positive and vimentin-negative, thus indicating that the responding cells were epithelial (figure 1). Checkerboard Analysis Checkerboard analysis demonstrated that the cell migration was due to both chemotaxis (directed migration) and chemokinesis (random migration) (table 1). The cell migration was measured in the presence of various dilutions ofmedium conditioned with bovine bronchial epithelial cells both above and below the filter membrane in order to make gradients of chemoattractant. Chemokinesis was present because even when no gradient existed, the number of cells that migrated increased as the concentration of conditioned medium increased. The cell migration was also chemotactic, however, because the number of cellsthat migrated increased when a gradient was present. Thus, bronchial epithelial cell migration in response to bronchial epithelial cell-conditioned medium was both chemokinetic and chemotactic. Dose Response of Epithelial Cell Migration The number of migrated epithelial cells decreased when the bronchial epithelial cell-conditioned medium was diluted with culture medium, indicating a dose dependence of the chemotactic activity. Cell migration with a chemotaxis membrane to which cellshad been preattached showed a similar dose dependence, indicating that the epithelial cell migration was not due to increased cell attachment (figure 2). Time-dependent and De Novo Production of Chemotactic Activity by Bovine Bronchial Epithelial Cells Bovine bronchial epithelial cells accumulated chemotactic activity in the supernatant medium with increasing time,
Fig. 1. Bronchial epithelial cells migrated through the chemotactic membrane. Top panel. Migrated bronchial epithelial cells stained by LeukoStat. Middlepanel. Migrated cells stained by antikeratin antibody. Bottom panel. Migrated cells stained by antivimentin antibody; magnification: x 400.
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AIRWAY EPITHELIAL CELL-DIRECTED EPITHELIAL CHEMOTAXIS
TABLE 1 CHECKERBOARD ANALYSIS OF CELL MIGRATION Dilution' Above Membranet
o Dilution below membrane:!:
o
3.0 3.3 10.7 62.0
1:4 1:2 1:1
± 0.6 ± 0.3 ± 0.7 ± 5.3
2.0 3.3 13.0 62.7
± 0.6 ± 0.3 ± 3.1 ± 0.7
1.7 1.7 8.3 44.7
± 0.3 ± 0.3 ± 2.3 ± 3.0
1.3 1.7 7.0 39.3
± 0.3 ± 0.7 ± 2.0 ± 2.3
, Ratio of dilution of crude bronchial epithelial cell-condilioned medium. 8ronchial epithelial cell suspension at 1 )( 10' cellslml in diluted conditioned medium. Diluted conditioned medium as a chemoattractant (number of migrated cells is indicated as average In triplicate ± SEM).
t
*
reaching a plateau at about three days. The addition of cycloheximide (25 ug/ ml) at the beginning of the incubation inhibited the release of chemotactic activity from bovine bronchial epithelial cells (figure 3). This suggests that the accumulation of chemotactic activity might represent de novo synthesis.
Partial Characterization of Chemotactic Activity The partial characterization of chemotactic activity in the medium conditioned with bovine bronchial epithelial cells revealed that the activity was nondialyza-
LL
ble, pepsin-labile, acid-stable, heat-labile, and lipid-inextractable (figure 4).
Inhibition of the Chemotactic Activity by Antifibronectin Antiserum Demonstration ofthe Molecular Size by The chemotactic activity of the gelatin0-150 Molecular Sieve Column binding portion was significantly inhibitChromatography ed by antifibronectin antiserum, and not To demonstrate the approximate molec- by normal rabbit serum. This confirms ular size of bovine bronchial epithelial that the gelatin-binding portion of checell-derived chemotactic activity, Sepha- motactic activity is fibronectin. Insulindex G-150 column chromatography was directed bronchial epithelial cell cheused. Chemotactic activity eluted as a sin- motaxis (5), however, was not inhibited gle sharp peak near the void volume of by antifibronectin antiserum, indicating the column. This peak coincided with the that inhibition of chemotaxis was not peak of fibronectin, which was measured a nonspecific effect of antifibronectin by ELISA (figure 5). antiserum (figure 7). Comparison of the Potency of Fibronectins from Different Sources as the Chemoattractants for Bronchial Epithelial Cells The chemotaxis assay using serially diluted fibronectins purified by gelatin-Sepharose 4B column chromatography from both bovine plasma and bovine epithelial cell-conditioned medium showed that locally produced fibronectin is more potent than plasma fibronectin as a chemoattractant. The difference is approximately 10- to 50-fold (figure 8).
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