Pulmonary Macrophages Can Stimulate Cell Growth of Bovine Bronchial Epithelial Cells Hajime Takizawa, Joe D. Beckmann, Shunsuke Shoji, Lorene R. Claassen, Ronald F. Ertl, James Linder, and Stephen I. Rennard Pulmonary and Critical Care Medicine Section, Department of Internal Medicine, and Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska
Macrophages are thought to participate in tissue repair following injury by releasing growth factors into the local environment. To evaluate whether pulmonary macrophages can mediate airway epithelial repair, we attempted to determine if pulmonary macrophages can stimulate growth of bovine bronchial epithelial cells in vitro. Bronchial epithelial cells isolated by protease digestion of the bovine bronchi were plated into tissue culture dishes with and without macrophage-conditioned medium. Bronchial epithelial cells cultured with macrophage-conditioned medium showed a significantly greater cell growth than those with.out macrophage-conditioned medium when assessed by direct enumeration of the cell numbers and by clonal growth assay. Stimulation of proliferation was confirmed by autoradiography using pH]thymidine uptake into cell nuclei. Co-culture of pulmonary macrophages with bronchial epithelial cells also led to an increase in cell number. Immunohistochemical staining of the proliferating cells showed that these cells were positively stained by anti-keratin antibodies confirming that they were bronchial epithelial cells. Partial characterization of the activity in macrophage-conditioned medium showed that it was nondialyzable, pepsin- and acid-labile, and lipid-inextractable. Sephadex 0-75 column fractionation indicated this activity existed in a high molecular fraction, thus suggesting a peptide. DEAE ion exchange chromatography revealed 3 peaks of stimulating activity. One peak resulted in a decrease in cell number, suggesting a possible inhibitory activity. The DEAE results thus suggest that macrophages may release several factors that can affect bronchial epithelial cell proliferation. In conclusion, pulmonary macrophages stimulate cell proliferation of bronchial epithelial cells in vitro. The stimulatory activity that may be heterogeneous appears to have the properties of a peptide.
The integrity of the bronchial epithelial mucosa is an important defense mechanism protecting against various kinds of noxious agents that are aspirated into airways. If the mucosal epithelial layer is injured, its recovery is largely dependent upon the replication of bronchial epithelial cells. These epithelial cells are composed of relatively slowly renewing populations in a normal state (1), but the rate of turnover is markedly enhanced by injury (2). Thus, the stimulation of replication of bronchial epithelial cells appears to be one response to injury of the bronchial structures that can occur in various pathologic processes. It has been suggested that macrophages might participate in the regulation of the repair
Key Words: pulmonary macrophage, bronchial epithelial cell, growthstimulating activity (Received in original form May 10, 1989 and in revised form October 16, 1989)
Address correspondence to: Hajime Takizawa, M.D., Pulmonary Section, Department of Internal Medicine, University of Nebraska Medical Center, 42nd and Dewey Avenue, Omaha, NE 68105. Abbreviations: avidin-biotin complex, ABC; bronchoalveolar lavage, BAL; bovine pituitary extract, BPE; RPM I 1640, RPMI. Am. J. Respir. Cell Mol. BioI. Vol. 2. pp. 245-255, 1990
processes following injury. Macrophages are one of the inflammatory cells that migrate and accumulate at a damaged area immediately after tissue injury. Moreover, these cells frequently persist until repair is complete (3). Macrophages have been reported to produce growth-stimulatory activity for mesenchymal cells including fibroblasts, smooth muscle cells, and endothelial cells (4-7). Recently it has also been reported that macrophages stimulate DNA synthesis of rat alveolar type II epithelial cells (8). In the present study, we attempted to determine if macrophages can stimulate the growth of bovine bronchial epithelial cells in vitro. The growth-stimulatory activity was assessed by direct enumeration of the cells, by clonal growth assay, by autoradiography, and by co-culture of bronchial epithelial cells with macrophages. These studies indicate macrophages can stimulate proliferation of bronchial epithelial cells. We have also performed partial characterization of this activity.
Materials and Methods Reagents LHC basal medium was purchased from Biofluids (Rockville, MD). HBSS, RPMI 1640 (RPMI), DMEM, MEM,
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HBSS containing 0.25 % trypsin, streptomycin-penicillin, and fungizone were purchased from GIBCO (Chagrin Falls, OH). FCS was obtained from Biofluids and heat-inactivated before use. Bacterial type XIV protease, transferrin, insulin, epidermal growth factor, and hydrocortisone were purchased from Sigma Chemical Co. (St. Louis, MO). Extraction of frozen bovine pituitaries from Pel Freez (Rogers, AR) was as previously described and yielded an extract containing 10 mg/ml protein (9). Bovine type I collagen (Vitrogen 100) was from Collagen Corporation (Palo Alto, CA). Isolation of Bovine Bronchial Epithelial Cells Bronchial epithelial cells were prepared by a modification of the method of Wu and colleagues (10, 11). Briefly, bovine lungs were obtained from a slaughterhouse just after killing. Bronchi were removed, cut into pieces, and trimmed of connective tissue. The bronchi were then incubated in sterile MEM that contained 0.1% protease, penicillin-streptomycin and fungizone at 4 0 C overnight. The bronchi were removed, and the bronchial lumens were gently rinsed several times with MEM containing 10% FCS to detach the epithelial cells. The collected cells were washed once with MEM with 10% FCS, filtered through 250-p.m sterile mesh (Tetko, Elmsford, Ny), and washed twice with MEM. The total cell count was calculated by a standard hemocytometer, and viability of the cells was examined by trypan blue dye exclusion. Approximately 500 X 106 to 1,000 X 106 cells (>80 to 98 % viability) were obtained from each lung. To confirm that the cells thus obtained were of epithelial origin, immunohistochemical studies using the avidin-biotin complex (ABC) method were performed with anti-keratin antibody (MAK-6; Triton, Alameda, CA). The cells were also stained with anti-macrophage antibody (MAC387; DAKO, Santa Barbara, CA), and less than 0.5% of the cells were weakly positive. The cells were then washed with non-serumcontaining medium and resuspended in DMEM for use. Culture of Bronchial Epithelial Cells In Vitro The bronchial epithelial cells were plated at 5 x 105 cells/dish on 35-mm tissue culture plates (Corning, Corning, NY) and incubated at 37° C, 5% CO 2 overnight. Then the plates were rinsed with DMEM, and the medium was changed for the experimental purposes. Attachment of the primary cells to the tissue culture plastic was 5 to 20 %. The standard medium used in this report was LHC-9 medium (9, 12). This was LHC basal medium supplemented with bovine insulin (5 p.g/ml), epidermal growth factor (5 ng/ml), bovine transferrin (10 p.g/ml), retinoic acid (33 nM), T 3 (10 nM), hydrocortisone (0.2 p.M), epinephrine (5 p.g/ml), phosphoethanolamine/ethanolamine (5 p.M), trace elements, calcium (0.11 mM), bovine pituitary extract (BPE, 0.5%), penicillinstreptomycin, and fungizone. The medium was changed every 3 d unless otherwise mentioned. Procedures to Obtain Bovine Pulmonary Macrophages and Macrophage-conditioned Medium Bronchoalveolar lavage (BAL) was performed with 10 ml sterile physiologic saline 10 to 15 times in each bovinebronchus to obtain macrophages. The average recovery of BAL fluid volume was above 80%, and the cell viability was above
95 % as measured by trypan blue exclusion. After washing twice with HBSS without calcium and magnesium, the cell number and viability were examined. The cells were also cytocentrifuged (Cytospin II; Shandon, Sewickley, PA) and stained by Leuko Stat stain (Fisher Scientific, Orangeburg, NY). The cells were also immunohistochemically stained by the ABC method with anti-human macrophage antibody (MAC387, DAKO) to identify macrophages. Cells were used only when the percentage of macrophages was no less than 95 % of the total cells. The cells were plated at 1 x 106 cells/ml in RPMI at 37° C, 5 % CO 2 • After 24 h, the supernatant medium was harvested and was stored at -80° C until direct use without dialysis unless otherwise mentioned. Similar experiments have been conducted in which the macrophages were cultured for 24 h in RPMI plus 0.5 % serum or LHC-9 media. Similar growth-stimulatory activity of the macrophage-conditioned media on the bronchial epithelial cells was observed in comparison to the results presented below. Supernatant media were also collected from macrophages that were cultured in RPMI with various kinds of stimuli: (1) LPS (5 p.g/ml) (Difco, Detroit, MI); (2) zymosan (2 mg/ml [1 ml of boiled, washed particles]) (Sigma) that was incubated with 10 ml of fresh bovine serum for 30 min at 37° C, washed, and suspended in RPMI; and (3) PMA (0.1 p.M) (Sigma). As a control, each stimulus was incubated in parallel under identical conditions without macrophages. After 24 h, the supernatant media were centrifuged and harvested. The conditioned media (10 ml) were dialyzed against 500 ml RPMI, filter-sterilized, and kept at -80 0 C until use. Quantification of the Cell Growth by Coulter Counter In order to determine if macrophage-conditioned medium would stimulate bronchial epithelial cell proliferation, bronchial epithelial cells were prepared and plated as described above. After 24 h for attachment, the plates were rinsed to remove the unattached cells and fresh media containing various concentrations of macrophage-conditioned media were added. As a control, the same percentage of unconditioned RPM I that had been incubated without macrophages was used. The cell number was then counted by Coulter counter (Coulter Electronics, Elk Grove Village, IL) after 7 d. To accomplish this, each plate was rinsed once with HBSS, HBSS containing 0.25 % trypsin was added, and the cells were detached from the plates. Each time after this procedure, it was confirmed that all of the cells were detached from the plates by direct inspection by phase-contrast microscopy. The cell count was performed twice for each of triplicate dishes. Cell Growth Curve With and Without Macrophage-conditioned Medium In order to determine the time course ofthe response ofbronchial epithelial cells to macrophage-conditioned medium and to test the requirement for added BPE, cells were prepared and plated in DMEM as described above. After 24 h, DMEM was replaced with LHC-9 supplemented with various concentrations of BPE containing 30 % macrophage-conditioned media or 30 % unconditioned RPMI. Cell counts were performed as described above after various periods of time.
Takizawa, Beckmann, Shoji et al.: Macrophages Stimulate Bronchial Epithelial Cell Growth
Morphologic and Immunohistochemical Evaluation of Responding Cells For in situ observation of the cell morphology during the cell culture, an inverted, phase-contrast microscope (Swift Instruments International, San Jose, CA) was used. Cells were stained with a modified Wright's stain (Leuko Stat; Fisher). To confirm that responding cells were of epithelial origin, immunohistochemical studies using the ABC method were performed with anti-keratin antibody (MAK-6; Triton) for epithelial cells. Effect of Macrophage-conditioned Medium on DNA Synthesis by Bronchial Epithelial Cells To demonstrate DNA synthesis, (3H]thymidine uptake to the cell nucleus during cell culture, followed by autoradiography, was performed. To accomplish this, the bronchial epithelial cells were plated at 5 X 104 cells/well onto Lab-Tek Chamber Slides (8-chamber; Nunc, Naperville, IL) precoated with Vitrogen 100 (30 j.tg/ml). After overnight incubation, unattached cells were removed and the media were changed to fresh media containing 30% macrophage conditioned media or 30% RPMI. After the cells were allowed to grow for 24 and 72 h, 5 j.tCi/ml [3H]thymidine (sp act; 15.0 Ci/mmol; New England Nuclear, Boston, MA) was added to the samples. After an incubation period of 18 to 20 h, the media were removed and [3H]thymidine pulse-labeled cells were fixed with methanol at room temperature. The slides were then coated with NTB-2 tracking emulsion diluted 1:2 with deionized water at 42° C, exposed at -20° C for 7 d, developed in Kodak D-19 developer for 3 min, and fixed in Kodak fixer for 6 min. The samples were stained thereafter with hematoxy lin-eosin. Autoradiographic grains within the nucleus could easily be visualized by light microscopy. The epithelial cells that contained more than 30 dots within the nucleus were scored as positive cells. The percentage of positive cells was determined by examining 300 cells in each chamber. Quantification of the Cell Growth by Clonal Growth Assay A further approach to the analysis of cell growth is the clonal growth assay as developed for keratinocytes (13). For these experiments, first-passaged cells were prepared. Primary cells on 100-mm tissue culture dishes (Corning) were incubated 10 to 14 din LHC-9 media. Prior to confluence, the cells were removed using I ml of 0.05% trypsin/I % polyvinylpyrolidone/0.5 mM EGTA (9) followed by addition of 0.1 ml soybean trypsin inhibitor (0.5%). Cells were gently dispersed by pipette, suspended in LHC-9, counted with a hemocytometer, and then plated in duplicate at 3 densities (see Figure 5) onto 35-mm wells of6-well plates (Cell Wells; Corning) that had been pre-incubated for I to 2 h with bovine type I collagen (Vitrogen; 30 j.tg/ml well). Attachment was usually 70 to 90% within I to 2 h. Media were changed the following day and every 2 to 3 d thereafter. After 9 d, plates were rinsed with PBS, fixed with methanol, and stained with Leuko Stat. The areas covered by cell colonies in each well were quantified by an Optomax V Image Analyzer (Optomax, Hollis, NH) equipped with a video camera and 35-mm
247
camera lens. We have observed a linear correlation of cell count measured by hemocytometer, with area measured as above (not shown). Co-culture of the Macrophages with Bronchial Epithelial Cells In order to confirm that macrophages could stimulate proliferation, co-culture experiments were performed. Primary bronchial epithelial cells were plated at 5 X 105 cells in 35-mm tissue culture plates. After overnight incubation at 37° C, 5 % CO 2 , unattached cells were removed and various concentrations of macrophages (>95 % purity) obtained by BAL were added to the dishes, and the mixture of cells co-cultured. Plates with bronchial epithelial cells alone and those with macrophages alone were simultaneously cultured as controls. The medium was LHC-9 minus BPE. Because preliminary studies have shown that some of the macrophages without FCS appeared to be dying after more than 36 h of culture, we added 0.5 % FCS to each dish of all groups in the co-culture experiments; with 0.5 % FCS, macrophages were viable by trypan blue dye exclusion technique (~ 96 %) after 7 d of culture. With that concentration of FCS, the bronchial epithelial cells did not show any significant cell growth as compared to media without FCS (data not shown). Toconfirm that the cells that proliferated in the co-culture experiment were epithelial cells, responding cells were studied with anti-keratin monoclonal antibody as described above. The cells Were also stained by the ABC method with an anti-human macrophage antibody, MAC387 (DAKO). Preliminary studies (not shown) demonstrated cross-reactivity of this antibody with bovine macrophages. Partial Characterization of the Activity Several treatments of macrophage-conditioned media were performed for evaluation of the biochemical characteristics of the activity. First, molecules less than 6,000 to 8,000 D were removed by dialysis (Spectrum, Los Angeles, CA) against two 50-fold exchange volumes of RPMI at 4°C for 12 h. Second, conditioned medium was incubated with 0.1 mg/ml pepsin (Sigma) for 2 h at 37° C after adjusting the pH to 2.5 with 0.5 M acetic acid. After digestion, the pH was adjusted to 7.4, and the sample was dialyzed against RPMI. Third, acid treatment was done by incubation of the sample with 0.5 M acetic acid at pH 2.5 at 37° C for 2 h. The sample was then adjusted to 7.4 and dialyzed against RPMI. Fourth, heat treatment was done by boiling the sample at 100° C for 15 min. Finally, lipid extraction was done by vortexing the sample with 2 volumes of ethyl acetate twice. The lipid phases were combined, dried under nitrogen, and resuspended in RPMI. The aqueous phase was also dried to remove the remaining ethyl acetate, then used as a sample. Partial Purification of the Growth-stimulatory Activity by Column Chromatography In order to determine the apparent molecular weight of the activity, the macrophage-conditioned medium was applied to a Sephadex G-75 column (55 em X 1.5 em) (Pharmacia, Piscataway, NJ). Each fraction of 5 ml was obtained and subjected to the bioassay for the activity as described above.
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Blue dextran (2 X lQ6 D), BSA (66,000 D), and phenol red (354 D) were used as molecular weight markers. Macrophage-conditioned medium was also applied to a DEAEcellulose column (24 em x 3 cm) (Pharmacia). The column was washed with 500 ml running buffer (20 mM Tris-HCl, pH 7.4, 20 mM NaCl) and then eluted with a linear 300-ml salt gradient (20 to 300 mM NaCl), followed by a 300-ml 2.0 M NaCI step. Sample fractions of 5 ml were dialyzed against 500 ml RPMI, diluted 1:10 in LHC-9 (0.2 % BPE), and then subjected to the bioassay as described above. Statistics Student's t test was used to compare the data of 2 experimental groups.
Results Stimulation of Bronchial Epithelial Cell Proliferation by Macrophage-conditioned Medium To evaluate the effect of the macrophage-conditioned media on the cell growth of bronchial epithelial cells in vitro, the bronchial epithelial cells were cultured in 35-mm culture dishes (5 X 105 cells/dish) with and without addition of various percentages of macrophage-conditioned media. The bronchial epithelial cells cultured in LHC-9 supplemented
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with 0 to 40% macrophage-conditioned media showed a dose-dependent stimulation of cell number when assessed by Coulter counter (Figure 1). Because 30 % conditioned medium in LHC-9 appeared to be an optimal concentration for the stimulation of bronchial epithelial cell growth, this percentage was used for further experiments. Our preliminary studies showed that the cell growth of bronchial epithelial cells was dependent on the percentage of supplemented BPE (0.1% was a threshold dose and 0.5 %
Takizawa, Beckmann, Shoji et al.: Macrophages Stimulate Bronchial Epithelial Cell Growth
Figure 3. Morphologic findings of the bronchial epithelial cells in culture. Top panel: a photomicrograph showing the in situ cell morphology after 7 d in culture. The cultured cells were polygonal (original magnification: x200). Middle panel: a photomicrograph of the specimen stained by anti-keratin antibody conjugated with biotin. The cultured cells were keratinpositive in their cytoplasms (7 d in culture) (original magnification: x400). Bottom panel: a photomicrograph of the preparation used for autoradiography. The small dots within the nuclei showed an uptake of [3H]thymidine corresponding to DNA synthesis ([3H]thymidine was added to the cells after 72 h of culture with macrophage-conditioned media) (original magnification: x400). All the samples are those from plates in which bronchial epithelial cells were cuhured with macrophage-conditioned media.
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was an optimal or saturating dose), as suggested previously (9, 11). Therefore, we evaluated the effect of macrophageconditioned medium in LHC-9 with 3 different concentrations ofBPE: 0,0.1, and 0.5% (Figure 2). As shown in Figure 2, bronchial epithelial cells in media supplemented with 30 % macrophage-conditioned media showed a significantly greater cell increase than those in media with 30 % RPMI alone (control) in each kind of medium. Although the percentage increase in cell number was greatest in the absence of BPE, the effects could be seen earlier in 0.5 % BPE. Morphologic changes of the cells during culture were investigated by phase-contrast microscopy. The cells were polygonal, and mitotic figures could be observed (Figure 3, top). Immunohistochemical staining demonstrated that the cells were keratin-positive, indicating they were of epithelial origin (Figure 3, middle). pH]thymidine uptake by the nuclei ofthe bronchial epithelial cells was assessed to further verify the stimulatory effect of macrophage-conditioned medium on DNA synthesis. The cells whose nucleus was positive were easily identified using light microscopy (as shown in Figure 3, bottom), and the percentage of the positive cells was quantified. The bronchial epithelial cells cultured with conditioned media showed a significant increase in percent (3H]-positive cells compared to those without macrophage-conditioned media (Student's t test, P < 0.01) (Figure 4). The growth-stimulatory activity of macrophage-conditioned media was also evaluated by clonal growth assay (see
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Macrophages are thought to participate in tissue repair following injury by releasing growth factors into the local environment. To evaluate whether pulmonary macrophages can mediate airway epithelial repair, we attempted to determine if pulmonary macrophages can stimulate growth of bovine bronchial epithelial cells in vitro. Bronchial epithelial cells isolated by protease digestion of the bovine bronchi were plated into tissue culture dishes with and without macrophage-conditioned medium. Bronchial epithelial cells cultured with macrophage-conditioned medium showed a significantly greater cell growth than those with.out macrophage-conditioned medium when assessed by direct enumeration of the cell numbers and by clonal growth assay. Stimulation of proliferation was confirmed by autoradiography using pH]thymidine uptake into cell nuclei. Co-culture of pulmonary macrophages with bronchial epithelial cells also led to an increase in cell number. Immunohistochemical staining of the proliferating cells showed that these cells were positively stained by anti-keratin antibodies confirming that they were bronchial epithelial cells. Partial characterization of the activity in macrophage-conditioned medium showed that it was nondialyzable, pepsin- and acid-labile, and lipid-inextractable. Sephadex 0-75 column fractionation indicated this activity existed in a high molecular fraction, thus suggesting a peptide. DEAE ion exchange chromatography revealed 3 peaks of stimulating activity. One peak resulted in a decrease in cell number, suggesting a possible inhibitory activity. The DEAE results thus suggest that macrophages may release several factors that can affect bronchial epithelial cell proliferation. In conclusion, pulmonary macrophages stimulate cell proliferation of bronchial epithelial cells in vitro. The stimulatory activity that may be heterogeneous appears to have the properties of a peptide.
The integrity of the bronchial epithelial mucosa is an important defense mechanism protecting against various kinds of noxious agents that are aspirated into airways. If the mucosal epithelial layer is injured, its recovery is largely dependent upon the replication of bronchial epithelial cells. These epithelial cells are composed of relatively slowly renewing populations in a normal state (1), but the rate of turnover is markedly enhanced by injury (2). Thus, the stimulation of replication of bronchial epithelial cells appears to be one response to injury of the bronchial structures that can occur in various pathologic processes. It has been suggested that macrophages might participate in the regulation of the repair
Key Words: pulmonary macrophage, bronchial epithelial cell, growthstimulating activity (Received in original form May 10, 1989 and in revised form October 16, 1989)
Address correspondence to: Hajime Takizawa, M.D., Pulmonary Section, Department of Internal Medicine, University of Nebraska Medical Center, 42nd and Dewey Avenue, Omaha, NE 68105. Abbreviations: avidin-biotin complex, ABC; bronchoalveolar lavage, BAL; bovine pituitary extract, BPE; RPM I 1640, RPMI. Am. J. Respir. Cell Mol. BioI. Vol. 2. pp. 245-255, 1990
processes following injury. Macrophages are one of the inflammatory cells that migrate and accumulate at a damaged area immediately after tissue injury. Moreover, these cells frequently persist until repair is complete (3). Macrophages have been reported to produce growth-stimulatory activity for mesenchymal cells including fibroblasts, smooth muscle cells, and endothelial cells (4-7). Recently it has also been reported that macrophages stimulate DNA synthesis of rat alveolar type II epithelial cells (8). In the present study, we attempted to determine if macrophages can stimulate the growth of bovine bronchial epithelial cells in vitro. The growth-stimulatory activity was assessed by direct enumeration of the cells, by clonal growth assay, by autoradiography, and by co-culture of bronchial epithelial cells with macrophages. These studies indicate macrophages can stimulate proliferation of bronchial epithelial cells. We have also performed partial characterization of this activity.
Materials and Methods Reagents LHC basal medium was purchased from Biofluids (Rockville, MD). HBSS, RPMI 1640 (RPMI), DMEM, MEM,
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HBSS containing 0.25 % trypsin, streptomycin-penicillin, and fungizone were purchased from GIBCO (Chagrin Falls, OH). FCS was obtained from Biofluids and heat-inactivated before use. Bacterial type XIV protease, transferrin, insulin, epidermal growth factor, and hydrocortisone were purchased from Sigma Chemical Co. (St. Louis, MO). Extraction of frozen bovine pituitaries from Pel Freez (Rogers, AR) was as previously described and yielded an extract containing 10 mg/ml protein (9). Bovine type I collagen (Vitrogen 100) was from Collagen Corporation (Palo Alto, CA). Isolation of Bovine Bronchial Epithelial Cells Bronchial epithelial cells were prepared by a modification of the method of Wu and colleagues (10, 11). Briefly, bovine lungs were obtained from a slaughterhouse just after killing. Bronchi were removed, cut into pieces, and trimmed of connective tissue. The bronchi were then incubated in sterile MEM that contained 0.1% protease, penicillin-streptomycin and fungizone at 4 0 C overnight. The bronchi were removed, and the bronchial lumens were gently rinsed several times with MEM containing 10% FCS to detach the epithelial cells. The collected cells were washed once with MEM with 10% FCS, filtered through 250-p.m sterile mesh (Tetko, Elmsford, Ny), and washed twice with MEM. The total cell count was calculated by a standard hemocytometer, and viability of the cells was examined by trypan blue dye exclusion. Approximately 500 X 106 to 1,000 X 106 cells (>80 to 98 % viability) were obtained from each lung. To confirm that the cells thus obtained were of epithelial origin, immunohistochemical studies using the avidin-biotin complex (ABC) method were performed with anti-keratin antibody (MAK-6; Triton, Alameda, CA). The cells were also stained with anti-macrophage antibody (MAC387; DAKO, Santa Barbara, CA), and less than 0.5% of the cells were weakly positive. The cells were then washed with non-serumcontaining medium and resuspended in DMEM for use. Culture of Bronchial Epithelial Cells In Vitro The bronchial epithelial cells were plated at 5 x 105 cells/dish on 35-mm tissue culture plates (Corning, Corning, NY) and incubated at 37° C, 5% CO 2 overnight. Then the plates were rinsed with DMEM, and the medium was changed for the experimental purposes. Attachment of the primary cells to the tissue culture plastic was 5 to 20 %. The standard medium used in this report was LHC-9 medium (9, 12). This was LHC basal medium supplemented with bovine insulin (5 p.g/ml), epidermal growth factor (5 ng/ml), bovine transferrin (10 p.g/ml), retinoic acid (33 nM), T 3 (10 nM), hydrocortisone (0.2 p.M), epinephrine (5 p.g/ml), phosphoethanolamine/ethanolamine (5 p.M), trace elements, calcium (0.11 mM), bovine pituitary extract (BPE, 0.5%), penicillinstreptomycin, and fungizone. The medium was changed every 3 d unless otherwise mentioned. Procedures to Obtain Bovine Pulmonary Macrophages and Macrophage-conditioned Medium Bronchoalveolar lavage (BAL) was performed with 10 ml sterile physiologic saline 10 to 15 times in each bovinebronchus to obtain macrophages. The average recovery of BAL fluid volume was above 80%, and the cell viability was above
95 % as measured by trypan blue exclusion. After washing twice with HBSS without calcium and magnesium, the cell number and viability were examined. The cells were also cytocentrifuged (Cytospin II; Shandon, Sewickley, PA) and stained by Leuko Stat stain (Fisher Scientific, Orangeburg, NY). The cells were also immunohistochemically stained by the ABC method with anti-human macrophage antibody (MAC387, DAKO) to identify macrophages. Cells were used only when the percentage of macrophages was no less than 95 % of the total cells. The cells were plated at 1 x 106 cells/ml in RPMI at 37° C, 5 % CO 2 • After 24 h, the supernatant medium was harvested and was stored at -80° C until direct use without dialysis unless otherwise mentioned. Similar experiments have been conducted in which the macrophages were cultured for 24 h in RPMI plus 0.5 % serum or LHC-9 media. Similar growth-stimulatory activity of the macrophage-conditioned media on the bronchial epithelial cells was observed in comparison to the results presented below. Supernatant media were also collected from macrophages that were cultured in RPMI with various kinds of stimuli: (1) LPS (5 p.g/ml) (Difco, Detroit, MI); (2) zymosan (2 mg/ml [1 ml of boiled, washed particles]) (Sigma) that was incubated with 10 ml of fresh bovine serum for 30 min at 37° C, washed, and suspended in RPMI; and (3) PMA (0.1 p.M) (Sigma). As a control, each stimulus was incubated in parallel under identical conditions without macrophages. After 24 h, the supernatant media were centrifuged and harvested. The conditioned media (10 ml) were dialyzed against 500 ml RPMI, filter-sterilized, and kept at -80 0 C until use. Quantification of the Cell Growth by Coulter Counter In order to determine if macrophage-conditioned medium would stimulate bronchial epithelial cell proliferation, bronchial epithelial cells were prepared and plated as described above. After 24 h for attachment, the plates were rinsed to remove the unattached cells and fresh media containing various concentrations of macrophage-conditioned media were added. As a control, the same percentage of unconditioned RPM I that had been incubated without macrophages was used. The cell number was then counted by Coulter counter (Coulter Electronics, Elk Grove Village, IL) after 7 d. To accomplish this, each plate was rinsed once with HBSS, HBSS containing 0.25 % trypsin was added, and the cells were detached from the plates. Each time after this procedure, it was confirmed that all of the cells were detached from the plates by direct inspection by phase-contrast microscopy. The cell count was performed twice for each of triplicate dishes. Cell Growth Curve With and Without Macrophage-conditioned Medium In order to determine the time course ofthe response ofbronchial epithelial cells to macrophage-conditioned medium and to test the requirement for added BPE, cells were prepared and plated in DMEM as described above. After 24 h, DMEM was replaced with LHC-9 supplemented with various concentrations of BPE containing 30 % macrophage-conditioned media or 30 % unconditioned RPMI. Cell counts were performed as described above after various periods of time.
Takizawa, Beckmann, Shoji et al.: Macrophages Stimulate Bronchial Epithelial Cell Growth
Morphologic and Immunohistochemical Evaluation of Responding Cells For in situ observation of the cell morphology during the cell culture, an inverted, phase-contrast microscope (Swift Instruments International, San Jose, CA) was used. Cells were stained with a modified Wright's stain (Leuko Stat; Fisher). To confirm that responding cells were of epithelial origin, immunohistochemical studies using the ABC method were performed with anti-keratin antibody (MAK-6; Triton) for epithelial cells. Effect of Macrophage-conditioned Medium on DNA Synthesis by Bronchial Epithelial Cells To demonstrate DNA synthesis, (3H]thymidine uptake to the cell nucleus during cell culture, followed by autoradiography, was performed. To accomplish this, the bronchial epithelial cells were plated at 5 X 104 cells/well onto Lab-Tek Chamber Slides (8-chamber; Nunc, Naperville, IL) precoated with Vitrogen 100 (30 j.tg/ml). After overnight incubation, unattached cells were removed and the media were changed to fresh media containing 30% macrophage conditioned media or 30% RPMI. After the cells were allowed to grow for 24 and 72 h, 5 j.tCi/ml [3H]thymidine (sp act; 15.0 Ci/mmol; New England Nuclear, Boston, MA) was added to the samples. After an incubation period of 18 to 20 h, the media were removed and [3H]thymidine pulse-labeled cells were fixed with methanol at room temperature. The slides were then coated with NTB-2 tracking emulsion diluted 1:2 with deionized water at 42° C, exposed at -20° C for 7 d, developed in Kodak D-19 developer for 3 min, and fixed in Kodak fixer for 6 min. The samples were stained thereafter with hematoxy lin-eosin. Autoradiographic grains within the nucleus could easily be visualized by light microscopy. The epithelial cells that contained more than 30 dots within the nucleus were scored as positive cells. The percentage of positive cells was determined by examining 300 cells in each chamber. Quantification of the Cell Growth by Clonal Growth Assay A further approach to the analysis of cell growth is the clonal growth assay as developed for keratinocytes (13). For these experiments, first-passaged cells were prepared. Primary cells on 100-mm tissue culture dishes (Corning) were incubated 10 to 14 din LHC-9 media. Prior to confluence, the cells were removed using I ml of 0.05% trypsin/I % polyvinylpyrolidone/0.5 mM EGTA (9) followed by addition of 0.1 ml soybean trypsin inhibitor (0.5%). Cells were gently dispersed by pipette, suspended in LHC-9, counted with a hemocytometer, and then plated in duplicate at 3 densities (see Figure 5) onto 35-mm wells of6-well plates (Cell Wells; Corning) that had been pre-incubated for I to 2 h with bovine type I collagen (Vitrogen; 30 j.tg/ml well). Attachment was usually 70 to 90% within I to 2 h. Media were changed the following day and every 2 to 3 d thereafter. After 9 d, plates were rinsed with PBS, fixed with methanol, and stained with Leuko Stat. The areas covered by cell colonies in each well were quantified by an Optomax V Image Analyzer (Optomax, Hollis, NH) equipped with a video camera and 35-mm
247
camera lens. We have observed a linear correlation of cell count measured by hemocytometer, with area measured as above (not shown). Co-culture of the Macrophages with Bronchial Epithelial Cells In order to confirm that macrophages could stimulate proliferation, co-culture experiments were performed. Primary bronchial epithelial cells were plated at 5 X 105 cells in 35-mm tissue culture plates. After overnight incubation at 37° C, 5 % CO 2 , unattached cells were removed and various concentrations of macrophages (>95 % purity) obtained by BAL were added to the dishes, and the mixture of cells co-cultured. Plates with bronchial epithelial cells alone and those with macrophages alone were simultaneously cultured as controls. The medium was LHC-9 minus BPE. Because preliminary studies have shown that some of the macrophages without FCS appeared to be dying after more than 36 h of culture, we added 0.5 % FCS to each dish of all groups in the co-culture experiments; with 0.5 % FCS, macrophages were viable by trypan blue dye exclusion technique (~ 96 %) after 7 d of culture. With that concentration of FCS, the bronchial epithelial cells did not show any significant cell growth as compared to media without FCS (data not shown). Toconfirm that the cells that proliferated in the co-culture experiment were epithelial cells, responding cells were studied with anti-keratin monoclonal antibody as described above. The cells Were also stained by the ABC method with an anti-human macrophage antibody, MAC387 (DAKO). Preliminary studies (not shown) demonstrated cross-reactivity of this antibody with bovine macrophages. Partial Characterization of the Activity Several treatments of macrophage-conditioned media were performed for evaluation of the biochemical characteristics of the activity. First, molecules less than 6,000 to 8,000 D were removed by dialysis (Spectrum, Los Angeles, CA) against two 50-fold exchange volumes of RPMI at 4°C for 12 h. Second, conditioned medium was incubated with 0.1 mg/ml pepsin (Sigma) for 2 h at 37° C after adjusting the pH to 2.5 with 0.5 M acetic acid. After digestion, the pH was adjusted to 7.4, and the sample was dialyzed against RPMI. Third, acid treatment was done by incubation of the sample with 0.5 M acetic acid at pH 2.5 at 37° C for 2 h. The sample was then adjusted to 7.4 and dialyzed against RPMI. Fourth, heat treatment was done by boiling the sample at 100° C for 15 min. Finally, lipid extraction was done by vortexing the sample with 2 volumes of ethyl acetate twice. The lipid phases were combined, dried under nitrogen, and resuspended in RPMI. The aqueous phase was also dried to remove the remaining ethyl acetate, then used as a sample. Partial Purification of the Growth-stimulatory Activity by Column Chromatography In order to determine the apparent molecular weight of the activity, the macrophage-conditioned medium was applied to a Sephadex G-75 column (55 em X 1.5 em) (Pharmacia, Piscataway, NJ). Each fraction of 5 ml was obtained and subjected to the bioassay for the activity as described above.
248
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Blue dextran (2 X lQ6 D), BSA (66,000 D), and phenol red (354 D) were used as molecular weight markers. Macrophage-conditioned medium was also applied to a DEAEcellulose column (24 em x 3 cm) (Pharmacia). The column was washed with 500 ml running buffer (20 mM Tris-HCl, pH 7.4, 20 mM NaCl) and then eluted with a linear 300-ml salt gradient (20 to 300 mM NaCl), followed by a 300-ml 2.0 M NaCI step. Sample fractions of 5 ml were dialyzed against 500 ml RPMI, diluted 1:10 in LHC-9 (0.2 % BPE), and then subjected to the bioassay as described above. Statistics Student's t test was used to compare the data of 2 experimental groups.
Results Stimulation of Bronchial Epithelial Cell Proliferation by Macrophage-conditioned Medium To evaluate the effect of the macrophage-conditioned media on the cell growth of bronchial epithelial cells in vitro, the bronchial epithelial cells were cultured in 35-mm culture dishes (5 X 105 cells/dish) with and without addition of various percentages of macrophage-conditioned media. The bronchial epithelial cells cultured in LHC-9 supplemented
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with 0 to 40% macrophage-conditioned media showed a dose-dependent stimulation of cell number when assessed by Coulter counter (Figure 1). Because 30 % conditioned medium in LHC-9 appeared to be an optimal concentration for the stimulation of bronchial epithelial cell growth, this percentage was used for further experiments. Our preliminary studies showed that the cell growth of bronchial epithelial cells was dependent on the percentage of supplemented BPE (0.1% was a threshold dose and 0.5 %
Takizawa, Beckmann, Shoji et al.: Macrophages Stimulate Bronchial Epithelial Cell Growth
Figure 3. Morphologic findings of the bronchial epithelial cells in culture. Top panel: a photomicrograph showing the in situ cell morphology after 7 d in culture. The cultured cells were polygonal (original magnification: x200). Middle panel: a photomicrograph of the specimen stained by anti-keratin antibody conjugated with biotin. The cultured cells were keratinpositive in their cytoplasms (7 d in culture) (original magnification: x400). Bottom panel: a photomicrograph of the preparation used for autoradiography. The small dots within the nuclei showed an uptake of [3H]thymidine corresponding to DNA synthesis ([3H]thymidine was added to the cells after 72 h of culture with macrophage-conditioned media) (original magnification: x400). All the samples are those from plates in which bronchial epithelial cells were cuhured with macrophage-conditioned media.
249
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was an optimal or saturating dose), as suggested previously (9, 11). Therefore, we evaluated the effect of macrophageconditioned medium in LHC-9 with 3 different concentrations ofBPE: 0,0.1, and 0.5% (Figure 2). As shown in Figure 2, bronchial epithelial cells in media supplemented with 30 % macrophage-conditioned media showed a significantly greater cell increase than those in media with 30 % RPMI alone (control) in each kind of medium. Although the percentage increase in cell number was greatest in the absence of BPE, the effects could be seen earlier in 0.5 % BPE. Morphologic changes of the cells during culture were investigated by phase-contrast microscopy. The cells were polygonal, and mitotic figures could be observed (Figure 3, top). Immunohistochemical staining demonstrated that the cells were keratin-positive, indicating they were of epithelial origin (Figure 3, middle). pH]thymidine uptake by the nuclei ofthe bronchial epithelial cells was assessed to further verify the stimulatory effect of macrophage-conditioned medium on DNA synthesis. The cells whose nucleus was positive were easily identified using light microscopy (as shown in Figure 3, bottom), and the percentage of the positive cells was quantified. The bronchial epithelial cells cultured with conditioned media showed a significant increase in percent (3H]-positive cells compared to those without macrophage-conditioned media (Student's t test, P < 0.01) (Figure 4). The growth-stimulatory activity of macrophage-conditioned media was also evaluated by clonal growth assay (see
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